J. Physiol. (1969), 203, pp. 337-357 337 With 9 text-figuras Printed in Great Britain

ACTIVITY OF IRRITANT RECEPTORS IN PULMONARY MICRO- EMBOLISM, ANAPHYLAXIS AND DRUG-INDUCED BRONCHOCONSTRICTIONS BY JANET E. MILLS, HILARY SELLICK AND J. G. WIDDICOMBE From the University Laboratory of Physiology, Oxford (Received 16 December 1968)

SUMMARY 1. Lung irritant receptors have been studied in rabbits by recording action potentials from single vagal nerve fibres. Some of the rabbits were bilaterally vagotomized, and some paralysed and artificially ventilated. 2. The receptors gave rapidly adapting irregular discharges on inflation and deflation of the . Many were stimulated by insufflation of am- monia vapour into the lungs, and some by passage of a fine catheter into the right bronchial tree. The fibres had conduction velocities in the range 3*6-25-8 m/sec. 3. The receptors were strongly stimulated by intravenous injections of histamine acid phosphate, 25-100 ,ug/kg. The response was considerably reduced by previous injection of isoprenaline which also reduced the due to histamine. 4. The receptors were stimulated by intravenous injections of isoprena- line, phenyl diguanide and micro-emboli, and by anaphylaxis induced in rabbits previously sensitized to egg albumin. 5. The receptor responses could not be closely correlated in size with simultaneous changes in total lung resistance, , or frequency. 6. It is concluded that, in rabbits with intact vagus nerves, lung irritant receptors contribute to the reflex hyperpnoea and bronchoconstriction of the conditions studied. INTRODUCTION A number of abnormal conditions in the lungs of experimental animals are associated with hyperventilation (increased minute volume and decreased PC02) and bronchoconstriction, both responses being prevented or greatly reduced by bilateral cervical vagotomy. These conditions include pulmonary micro-embolism (Megibow, Katz & Fernstein, 1943; Binet & 338 JANET E. MILLS AND OTHERS Burstein, 1946; Whitteridge, 1950; Niden & Aviado, 1956; Weidner & Light, 1958; Cahill, Attinger & Byrne, 1961; Halmagyi & Colebatch, 1961); anaphylaxis (Auer & Lewis, 1910; Karczewski & Widdicombe, 1969c); the responses to intravenous injections of histamine (Letona, Mata & Aviado, 1961; DeKock, Nadel, Zwi, Colebatch & Olsen, 1966; Nadel, Corn, Zwi, Flesh & Graf, 1966; Karczewski & Widdicombe, 1969b); and to phenyl diguanide (Dawes, Mott & Widdicombe, 1951; Barer & NUsser, 1953; Karczewski & Widdicombe, 1969b); and the of irritant gases (Cromer, Young & Ivy, 1933; Banister, Fegler & Hebb, 1950; Balchum, Dybicki & Meneely, 1960; Nadel, Salem, Tamplin & Tokiwa, 1965). A possible vagal afferent pathway for the reflex changes has been studied by Paintal (1955, 1957), who recorded action potentials in fine fibres from 'deflation receptors' in the cat. These receptors were stimulated by phenyl diguanide and by pulmonary micro-embolism; histamine, anaphylaxis and irritant gases were not tried. We have studied activity in vagal afferent fibres which appear to come from lung epithelial 'irritant receptors', with properties quite different from the deflation receptors described by Paintal (1955, 1957). This paper describes the responses of these irritant receptors in rabbits to pulmonary micro-embolism, anaphylaxis, phenyl diguanide, histamine and the inhalation of ammonia, and presents evidence that the receptors are an important component in the reflex effects ofthese conditions. Wehave used rabbits because they are relatively easy (compared with cats) to sensitize to foreign protein for later production of the anaphylactic reaction, and because their lung respiratory reflexes are stronger than those in most other mammalian species (Troelstra, 1960; Widdicombe, 1964). A preliminary report of some of these results has been given (Mills, Sellick & Widdicombe, 1969).

METHODS Twenty-four rabbits were anaesthetized with pentobarbitone sodium (Nembutal, Abbott), 32-50 mg/kg intravenously. Five cats were anaesthetized with pentobarbitone sodium, 32 mg/kg intraperitonally. Tracheal cannulae were inserted and also femoral venous catheters for injections of drugs. The animals were supine. Blood pressure was recorded from the left femoral artery through a polyethylene catheter by a capacitance manometer (Southern Instruments). The undamped natural frequency of the manometer and catheter system was in the range 20-28 c/s, assessed by the method of Hansen (1949). Injection of emboli was through a polyethylene catheter previously inserted through the right external jugular vein until pressure records typical of the right atrium were obtained; the same catheter was used in some experiments for recording right atrial pressure, with a capacitance manometer (Southern Instruments). Transpulmonary pressure was measured from an air- filled polyethylene cannula tied into a lower right anterior intrapleural space, and from a wide-bore needle inserted through the rubber-tube connexion to the tracheal cannula, using a differential capacitance manometer (Southern Instruments), range ± 50 cm H20. Tidal LUNG IRRITANT RECEPTORS 339 volume was measured from a Fleisch pneumotachograph 'head', linear range 0-110 ml./sec, connected to an inductance differential pressure recorder and integrator (Godart). End-tidal C02 % was measured with an infra-red absorption meter (Beckman-Spinco LBI), with a micro-sampling analysis cell connected distal to the pneumotachograph 'head' and sampling gas at about 300 ml./min; values were read from the meter. Action potentials were recorded from the distal end of the cut right cervical vagus nerve. The nerve was placed in a trough (3 x 2 x 2 cm deep) containing paraffin, and 'single-fibre' preparations were made. Platinum electrodes and conventional amplifiers (Tektronix) were used. Action potentials were also recorded in cats from tracheal efferent fibres with activity characteristic of vagal tracheobronchoconstrictor nerves (Widdicombe, 1961 a, 1966). Records of systemic arterial blood pressure, right atrial blood pressure, tidal volume, transpulmonary pressure and action potentials were displayed on an oscilloscope (Tektronix 551) and photographed on 7 cm paper (with a modified Cossor camera). The photographic record was analysed after enlargement 3 to 4 times by a projector (designed and built by Dr E. H. J. Schuster). In some experiments the variables were recorded on a Honeywell UV-31 oscillograph. Lung conductance and compliance were measured by the subtracterr' method of Mead & Whittenberger (1953), somewhat modified (Nadel & Widdicombe, 1962; Green & Widdi- combe, 1966). This involves displaying the component of transpulmonary pressure required to overcome resistance to flow and tracheal airflow on the two axes of an oscilloscope, to give 'loops', the slopes of which are proportional to total lung resistance, or its reciprocal, conductance (Karczewski & Widdicombe, 1969 a). Transpulmonary pressure, airflow and action potentials were also recorded on a four- channel FM tape recorder (Thermionix T 1000). This allowed re-evaluation of total lung resistance and lung compliance after the experiment had been completed. Anesthetized rabbits were paralysed by intravenous injections of gallamine triethiodide (10 mg), the injections being repeated if the animals made respiratory movements. The pump frequency was 20-30 inflations/min, and the tidal volume was adjusted to maintain end-tidal C02 % close to that during spontaneous breathing before paralysis (usually 3-4 %). Conduction velocities were measured by implanting a pair of platinum wire stimulation electrodes round the right vagus nerve in the upper thorax, and then closing the chest. In some experiments the right vagus nerve below the hilum of the lung was cut during the thoracostomy; this eliminated activity in afferent fibres from the abdominal viscera. Ammonia vapour was administered by injecting about 2-5 ml. air containing ammonia into the tracheal connexions during inspiration or pump inflation. The gas was taken from the gas phase in a conical flask containing ammonia solution. Mechanical stimulation of air- way receptors was performed by passing a 0 5 mm diameter nylon fibre or a 1F0 mm poly- ethylene tube through the tracheal cannula into the right bronchial tree. Nine rabbits were first sensitized to bovine serum albumin (Armour), by administration of three intraperitoneal doses of 0 1 ml. of a 20 % solution at 2-day intervals. After an interval at 3-6 weeks the rabbits were used for experiments, with 0-2 ml. of a 20 % solution of albumin as the challenging dose. Starch emboli were prepared by scraping fresh potato into 0 9 % saline (w/v), filtering the mixture through five layers of surgical gauze, and collect- ing the sediment. Volumes of diluted sediment (0 2-10 ml.) were injected. The maximum diameters of the starch particles were in the range 8-80 ,t, the majority being 20-30 ,u. Barium sulphate emboli (maximum crystal diameter, 20 ,y) were prepared from commercial barium sulphate (B.D.H.); 0 2-1 0 ml. volumes of a 33 0 (w/v) solution in 09 %0 saline were injected. Doses of histamine acid phosphate and of isoprenaline sulphate are expressed as the salts. Impulse frequencies were measured over respiratory cycles or pump ventilation cycles, since the receptors frequently had respiratory rhythms. The frequencies were then cal- culated as impulses/sec. For control values of impulse frequencies and of other variables, 340 JANET E. MILLS AND OTHERS measurements usually over at least five cycles were averaged. Maximum responses are given, as means of measurements over one to five cycles (usually three to five), depending on the pattern, regularity and duration of the response.

RESULTS Identification of pulmonary 'irritant' receptors. Epithelial 'irritant' receptors in the trachea and extrapulmonary bronchi ofthe cat are charac- terized by rapidly adapting discharges on constant volume infliations or deflations (or both) of the lungs or airways, and often by stimulation by irritant gases such as ammonia or sulphur dioxide; their fibres give large action potentials typical of myelinated fibres, with conduction velocities in the range 16-37 m/sec (Knowlton & Larrabee, 1946; Paintal, 1953; Widdicombe, 1954b). Histologically similar end-organs are seen in the epithelium both of the extrapulmonary and of the intrapulmonary air- ways. We therefore thought it likely that irritant receptors in the lungs would have similar properties to those outside the lungs. All vagal nerve strands were therefore tested for their responses to maintained large inflations and deflations; if rapidly adapting discharges were heard the strands were further dissected to give 'single-fibre' preparations. Many of these were then tested by insufflation of ammonia vapour, by mechanical stimulation with a fine plastic catheter or fibre introduced into the right bronchial tree, and by measurements of conduction velocity. If (a) the receptor gave a rapidly adapting irregular discharge to maintained large inflations and/or deflations, (b) the fibre was myelinated (Group A) on the evidence of conduction velocity or size of action potential, and (c) the receptor was not stimulated by an intra-luminal catheter inserted into the trachea and extrapulmonary bronchi (which would stimulate extrapul- monary 'cough receptors' (Widdicombe, 1954b)), the fibre was regarded as coming from a lung irritant receptor. The classification was reinforced if the receptor was stimulated by ammonia and by an intra-luminal catheter inserted deep into the right lung. Stimulation by intravenous injections of histamine acid phosphate was regarded as good supplementary evidence. The criteria will be considered further in the Discussion. Forty-four irritant receptors in twenty-six rabbits were studied. Responses to inflation and deflation. Of the forty-four receptors, thirty- six had their responses to maintained inflations and deflations ofthe lungs filmed and analysed. All gave rapidly adapting discharges similar to those of epithelial receptors in the walls of the extrapulmonary airways of the cat (Knowlton & Larrabee, 1946; Widdicombe, 1954b). Twelve receptors were more sensitive to inflation, twelve more sensitive to deflation, and twelve approximately equi-sensitive. Several receptors showed clear cardiac modulation of discharge on deflation of the lungs, possibly because LUNG IRRITANT RECEPTORS 341 the tissue containing the end-organ was pulled close to the beating heart. A response to inflation and deflation is illustrated in Fig. 1. iI = 00 +1 oE- PTp(20 cm -100 . fill I .I I I FIIwiifIt111l

I SEC Fig. 1. Responses of a pulmonary irritant receptor to deflation (upper record) and inflation (lower record) of the lungs. From above down: systemic arterial blood pressure (B.P.), tidal volume changes (VT, trace zeroing at points of zero airflow; inflation upwards), transpulmonary pressure (PT and action potentials in a single vagal fibre. Deflation and inflation were during the horizontal signal bars. Note the rapidly adapting irregular discharges. In this and other Figures absolute values of transpulmonary pressure are not given since it was used primarily for computing total lung resistance and compliance. Responses to epithelial irritation. Thirteen receptors, all in bilaterally vagotomized rabbits, were tested by insulation of 2-5 ml. air containing ammonia gas into the tracheal cannula connexions during spontaneous inspiration or pump-induced inflation of the lungs. Eight were stimulated, two slightly inhibited and three showed no change in discharge. For all thirteen fibres the control discharge frequency and increase in frequency were 4-7 + 3-0 and + 22-3 + 10-2 impulses/sec respectively (means and S.E., P 0.05). The latency of response was usually less than 2 sec (Fig. 2). With one exception, ammonia caused no measurable change in total lung resistance or compliance during the eight positive receptor responses. Most of the forty-four receptors were tested by passage of a fine (0.5-. 1*0 mm diameter) polyethylene catheter or nylon fibre; seven were vigorously stimulated and localized at depths of 13-4, 11-5, 10-7, 9, 6.5, 5 and 4 cm from the tracheostomy opening. These depths were shown by post mortem measurements to be beyond the tracheal bifurcation and in the airways of the right lung. The absence of a response in the majority of instances was taken as evidence that the receptors were not in the epithe- lium of the trachea and extrapulmonary bronchi, but in the intrapul- monary bronchi in sites not reached by the catheter; this conclusion was based on the fact that extrapulmonary epithelial receptors are very sensi- 342 JANET E. MILLS AND OTHERS tive to intraluminal mechanical stimulation (Widdicombe, 1954b). The responses to mechanical and chemical irritation were rapidly adapting, either of brief (1-3 see) total duration, or more usually with transient (0-2-1 0 see) bursts of activity separated by pauses (Fig. 2).

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1 sec Fig. 2. Response of a pulmonary irritant receptor to insufflation of ammonia vapour in a vagotomized, paralysed, artificially ventilated rabbit. From above down: tidal volume (VT), systemic arterial blood pressure (B.P.) and action potentials in a single vagal fibre. The two records are continuous. Ammonia gas was added at the signal mark during a lung inflation and caused an immediate irregular discharge. Total lung resistance and compliance were unchanged through- out the record. Conduction velocities. Conduction velocities were measured for six fibres, with values of 25-8, 18x0, 17*5, 7.5, 4-9 and 3-6 m/sec (mean, 12-9 m/sec). Responses to intravenous injections of histamine. All receptors were tested by intravenous injections of histamine acid phosphate, 50 /ug/kg. If there was no stimulation or a weak one, the dose was doubled; if the response was very vigorous, 25,ulg/kg was tried. These doses cause clear but not excessive increases in total lung resistance in anaesthetized rabbits with both cervical vagi intact (Karczewski & Widdicombe, 1969b; see also Discussion). Table 1 summarizes the results for twenty receptors in three conditions; spontaneously breathing with left vagus intact, when the histamine might cause vagal reflex changes in the pattern of breathing; spontaneously breathing with both vagi cut, when the pattern of breathing does not usually change (Karczewski & Widdicombe, 1969b); and during paralysis and artificial ventilation with both vagi cut, when the pattern of ventilation would be virtually constant. In all three conditions the receptor discharge significantly increased (Fig. 3), as did also total lung resistance, and lung compliance decreased. In only one of the thirty-six tests with histamine summarized in Table 1 did a receptor fail to increase its dis- LUNG IRRITANT RECEPTORS 343 o i * NC 0** 0 0 * *,I . . . . cm bo 14 +1 +1 +1 +1 +1 +10 c) 0 00"t In 0

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0 00 o_ COCOq4 344 JANET E. MILLS AND OTHERS charge frequency. (The means of the few receptor results in paralysed rabbits may be weighted by a relatively large number of tests with the largest dose of histamine.) The increases in receptor discharge seldom showed a close correlation in time with the changes in resistance, com- pliance, tidal volume, breathing frequency, transpulmonary pressure, right atrial or arterial blood pressure (Fig. 3). Nearly always the receptor in- creased firing before the change in lung mechanics. The mean latency for the receptor response was 7*1 + 4-2 see from injection. The first detectable

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I sec Fig. 3. Response of a pulmonary irritant receptor to intra-right atrial injection of histamine acid phoshpate, 100 /ig/kg (at signal in uppermost record) in a vagoto- mized, paralysed, artificially ventilated rabbit. Traces as in Fig. 2, 5*5 sec between upper two traces, 20-5 sec between lower two. Histamine caused an increase in blood pressure and a receptor discharge without clear relation to respiratory phase. Total lung resistance during the three records respectively was: uppermost, 0-32 cm H20/l./min; middle (at signal), 0-37 cm H20/l./min; and lowest, 0 77 cm H20/1./ min. Compliance was 4-83 ml./cm. H20 throughout. change in total lung resistance was usually about 10-20 see from the in- jection. However, it was difficult to time this accurately since resistances were averaged over each ventilation cycle and the onset of a change in resistance was gradual. The receptor discharge decreased towards control values while the changes in lung mechanics persisted. For five receptors the responses to identical doses ofhistamine were com- pared before and after left vagotomy, the right nerve being already cut. LUNG IRRITANT RECEPTORS 345 The nerve section diminished both the receptor response (but not signi- ficantly) and also the changes in total lung resistance and compliance. In addition to the results reported above, in further experiments twenty- four other receptors were tested with 25-100,ug/kg of the drug, nearly all in spontaneously breathing bilaterally vagotomized rabbits. Of these receptors, twenty were clearly stimulated, but- the results have not been analysed in detail, since the drug was used primarily to confirm the identity of the receptors. 200 I-O 100 E

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1sec Fig. 4. Response of a single tracheal efferent (constrictor) fibre to intravenous injection of histamine acid phosphate, 50 #cg/kg, in a paralysed, artificially venti- lated cat with intact cervical vagi. From above down: systemic arterial blood pressure, tidal volume and action potentials. Fifteen sec between upper two records, 14 see between lower two. Histamine was injected intravenously at the signal in the uppermost record, and caused a considerable increase in impulse traffic. Since the reflex effects of stimulation of the receptors probably include a vagal bronchoconstriction (see Discussion), we studied the action of hista- mine acid phosphate (50 jg/kg) on tracheal efferent fibres with activity characteristic of vagal tracheo-bronchoconstrictor nerves (Widdicombe, 1961a, 1966). These experiments had to be done in cats, since it has not 346 JANET E. MILLS AND OTHERS proved possible to record from such fibres in rabbits. In seven tests on five 'single-fibre' preparations the efferent activity increased (Fig. 4), the mean control and increase in discharge being 4-8 + 1-67 and + 7-2 + 2-45 impulses/ sec respectively (P < 0 05 for mean increase in discharge). Five of the seven tests were on paralysed and artificially ventilated animals. To test if the histamine-induced discharge from the irritant receptors was due to, or was being modified by the changes in bronchomuscular tone, in thirteen tests on six receptors the same dose of histamine was injected before and 20-60 see after an intravenous injection ofisoprenaline sulphate,

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LJ I sec Fig. 5. Response of a single pulmonary irritant receptor to intra-right atrial in- jection of histamine acid phosphate, 100 /ctg/kg (at double signal in uppermost record), about 30 see after intravenous injection of 50 ,tg isoprenaline sulphate. The rabbit and receptor were the same as those for Fig. 3, but in this instance the isoprenaline limited the receptor response to a few impulses in the middle record. 4 sec between upper two records, 20 see between lower two. Total lung resistance and compliance were unchanged during the three records at 0 57 cm H20/l./min and 4-42 ml./cm H20 respectively. 25 (eight experiments), 50 (four) or 100 tg (one). All the rabbits were bilaterally vagotomized; five tests were done in rabbits spontaneously breathing, and eight in animals paralysed and artificially ventilated. The results, which were similar in both groups of rabbits, are summarized in Table 1 and illustrated in Figs. 3 and 5. The histamine-induced increases LUNG IRRITANT RECEPTORS 347 in receptor discharge and in total lung resistance, and the decreases in lung compliance, were all significantly reduced by the isoprenaline. Control experiments established that sufficient time (10-15 min) had been left between the injections of histamine to rule out tachyphylaxis to the drug. Isoprenaline itself caused a significant but small increase in receptor discharge and an increase in total lung resistance (Table 2), but these effects had partially worn off by the time the second dose of histamine was injected. Similar responses to isoprenaline were obtained both in spon- taneously breathing and in paralysed, artificially ventilated rabbits. The receptor firing increased after isoprenaline in all of fourteen tests. 00 200w- md*IE 100t 1+1X_ S _,D~~~~~~~oC~~~~~~~-1X,

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1 sec Fig. 6. Response of a single pulmonary irritant receptor to intravenous injection of 100 jg phenyl diguanide (at signal in uppermost record) in a spontaneously breathingrabbitwith left cervicalvagus intact. Traces as in Fig. 1. Five sec between upper two records, 12 sec between lower two. Note that the stimulation of the receptor continues in the lowest record when the transpulmonary pressure swings had decreased to below the control valves. Responses to intravenous injections ofphenyl diguanide. In fourteen tests on twelve fibres the responses to intravenous injection of 100-150 g phenyl diguanide were studied. This stimulates pulmonary 'deflation' receptors (Dawes et al. 1951; Paintal, 1955) and part at least of its reflex respiratory action is via vagal fibres other than those stimulated by hista- mine (Karczewski & Widdicombe, 1969b). Table 2 summarizes the results, 348 JANET E. MILLS AND OTHERS

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B LUNG IRRITANT RECEPTORS 349 and Fig. 6 illustrates one experiment. Nine tests were in spontaneously breathing rabbits (five with the left vagus intact and four with both vagi cut); the remaining four tests were in bilaterally vagotomized, paralysed and artificially ventilated rabbits. Phenyl diguanide increased receptor discharge and total lung resistance in all three groups of rabbits. One experiment has been excluded from the calculation in Table 2; this receptor was inhibited by phenyl diguanide at the same time as the rabbit developed a periodic pattern of breathing, both responses being unique and un- explained. Of the remaining thirteen tests, in three instances there was no increase in receptor discharge after injection of phenyl diguanide. As with the histamine experiments there was no correlation in time between the increase in receptor discharge and the other variables measured (Fig. 6).

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I sec Fig. 7. Response of a single pulmonary irritant receptor to anaphylaxis induced by intra-right atrial injection of 0-2 ml. 20 % bovine serum albumin (at signal in upper record) in a previously sensitized rabbit, spontaneously breathing and bilaterally vagotomized. Traces, from above down: systemic arterial blood pres- sure, right atrial pressure (R.A.P. interrupted during injection), tidal volume, transpulmonary pressure and vagal impulses. Fifteen sec between the two records. Total lung resistance and compliance during the two records respectively were: upper 0 27 cm H20/l./min and 4-32 ml./cm H20; lower, 0-36 cm H20/l./min and 3-80 ml./cm H20. Responses during analyphliaxs. Nine receptors, in rabbits previously sensitized to bovine serum albumin, were studied during induction of anaphylactic shock by a further injection of albumin. All but one of the experiments were with bilaterally vagotomized spontaneously breathing animals. All experiments showed an increase in receptor discharge and an increase in total lung resistance (Table 2, Fig. 7). The mean receptor latency was 14 + 3-83 see from injection, and that for change in total lung resistance was 23+ 6-28 (P > 0-3 for mean difference). The receptor 350 JANET E. MILLS AND OTHERS responses did not correlate closely in time with changes in the other variables measured. Responses during pulmonary micro-embolism. In seven tests on five receptors, the responses to intravenous injections of small emboli (three with starch and four with barium sulphate) were studied. Four of the rabbits were bilaterally vagotomized. In all instances the receptor dis- charge increased (Table 2) and this occurred before any detectable change

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EI se Fig. 8. Response of a single pulmonary irritant receptor to intra-right atrial injection of 0 5 ml. barium sulphate emboli (at signal in uppermost record) in a bilaterally vagotomized, paralysed, artificially ventilated rabbit. Traces from above down: systemic arterial blood pressure, transpulmonary pressure, tidal volume and vagal impulses. First two records continuous; 10 sec between lower two. Total lung resistance and compliance during the three records respectively were: upper- most, 1-55 cm H20/l./min and 0-65 ml./cm H20; middle, 1-55 cm H20/1./min and 0-65 ml./cm H20; and lowest, 1-45 cm H20/l./min and 0-69 ml./cm H20. in lung mechanics or breathing. In four experiments the rabbits subse- quently died as the result of the embolism, but the increase in receptor firing took place before cessation of breathing or circulatory failure. Figure 8 illustrates an experiment. LUNG IRRITANT RECEPTORS 351 Progressive changes in 'control' impulse frequency. For the majority of receptors we noticed that the 'control' spontaneous discharge frequency increased progressively du-ring the course of an experiment with repeated injections of drugs and other tests. Figure 9 illustrates this effect for two receptors, one showing a considerable increase in spontaneous discharge and the other a smaller, more representative change in activity. These changes may have been associated with slow deterioration of the rabbits' haemodynamic and pulmonary conditions due to repeated testing.

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DISCUSSION The receptors formed a uniform group on the basis of (1) rapidly adapting responses to maintained inflations and/or defiations; (2) action potentials typical of small myelinated fibres (based upon comparison with action potentials from slowly adapting pulmonary stretch receptors), evidence supported by conduction velocity measurements; (3) irregular discharge pattern, often unrelated to respiratory phase during strong stimulation; (4) stimulation of some by an intrabronchial nylon fibre, which would enter only a small proportion of the right intrapulmonary 352 JANET E. MILLS AND OTHERS bronchial tree; (5) stimulation by ammonia vapour; and (6) increases in activity during anaphylaxis and micro-embolism, and after intravenous injections of histamine and phenyl diguanide. These properties distinguish them, first, from the pulmonary stretch receptors which mediate the Hering-Breuer inflation reflex. Such receptors are slowly adapting to maintained lung inflations, and usually do not in- crease their activity on lung deflations (Adrian, 1933; Knowlton & Larrabee, 1946; Widdicombe, 1954b). Some increase their discharge on inhalation ofirritant gases or after intravenous injections ofhistamine, but only weakly, with a regular discharge showing respiratory phase and, in the case of histamine, only if lung compliance decreases (Widdicombe, 1954c, 1961b; Whitteridge & Bfilbring, 1944). Their conduction velocities are in the range 14-59 m/sec in the cat (Paintal, 1953). They do not in- crease their discharge after multiple pulmonary micro-embolism (Walsh, 1946) or after intravenous injections of phenyl diguanide (Dawes et al. 1951). The reflex action of pulmonary stretch receptors is hypoventilation (Ramos, 1956; Lim, Luft & Grodins, 1958) and bronchodilatation (Loof- bourrow, Wood & Baird, 1957; Widdicombe & Nadel, 1963) and therefore cannot explain the vagal reflex effects of the lung conditions described in this paper. Secondly, the properties of the irritant receptors distinguish them from the deflation receptors studied by Paintal in the cat (1955, 1957). The latter end-organs are stimulated only weakly and briefly by lung deflation, and are inhibited by inflation of the lungs. They have fine vagal fibres, originally given conduction velocities in the range 3-9 m/sec (Paintal, 1953) but later said to correspond to non-myelinated fibres (Paintal, 1964). They lie deep in the lungs distal to the respiratory bronchioles (Paintal, 1957), and have regular discharges when stimulated by chemicals such as phenyl diguanide. The studies of pulmonary stretch receptors and of deflation receptors have been mainly with cats. However, it is unlikely that receptors and vagal fibre characteristics differ greatly between cat and rabbit. During this research we have recorded from fibres from pulmonary stretch receptors and from deflation receptors in the rabbit (unpublished), and other authors also have described the properties of fibres from pulmonary stretch receptors (Weidmann, Berde & Bucher, 1949) and from deflation receptors (Homberger, 1968) in rabbits. All these results indicate that there are no qualitative differences between cat and rabbit with regard to these two groups of receptors. Homberger (1968) has recorded from vagal fibres probably from pulmonary irritant receptors in the rabbit, but she has not studied their responses in the conditions described in this paper. The evidence that the fibres come from lung epithelial irritant receptors LUNG IRRITANT RECEPTORS 353 is as follows. (1) Their properties are qualitatively identical to those of irritant receptors which have been unequivocally localized in the epithe- lium of the trachea and extrapulmonary bronchi of the cat (Widdicombe, 1954b). Thus receptors of both groups are characterized by rapid adapta- tion to inflation and/or deflation, connexion to myelinated afferent vagal fibres, mechanical stimulation by epithelial contact with a catheter, stimulation by inhalation of chemical irritants, and irregular discharge of action potentials at high frequency. In addition some of the tracheal receptors are stimulated by intravenous injections of histamine (J. A. Nadel & J. G. Widdicombe, unpublished). (2) Histological evidence sug- gests that the only pulmonary receptors which have myelinated afferent fibres and are appropriately situated to respond to mechanical and chemical irritation are sub- or intra-epithelial endings (e.g. Larsell, 1921; Elftman, 1943; Widdicombe, 1964). In the remainder of the discussion we will assume that the fibres from which we have recorded were arising from epithelial irritant receptors in the intrapulmonary airways. Most of the receptors had no or little spontaneous discharge in eupnoeic rabbits, but fired with inspiratory rhythm after bilateral vagotomy. This is not surprising since they usually responded to deep inflations and vagotomy caused deep breathing. The progressive increase in 'control' discharge rate seen during the course of a long period of recording (Fig. 9) may correspond to slow development of atelectasis or pulmonary inter- stitial oedema due to the repeated tests being conducted; accumulation of might also play a part. The receptors discharged more when the rabbits breathed deeper, and might be expected to fire more if lung compliance decreased, since there would be a greater elastic pull on the airways (Severinghaus & Stupfel, 1955; Caro, Butler & IDubois, 1960). However, these mechanisms cannot explain the changes in discharge in response to any of the tests used. The positive receptor responses were present in paralysed artificially ventilated rabbits, there was seldom measurable decrease in compliance when the receptors first increased their activity, and the discharge usually lacked or lost any respiratory phase. Similarly, the receptor responses could seldom be correlated with changes in total lung resistance, and often occurred in their absence. A lack of correlation between mean increases in receptor discharge and mean changes in total lung resistance and lung compliance can be seen in the results given in Tables 1 and 2. The fact that the hista- mine-induced receptor discharge was prevented by isoprenaline is therefore unlikely to be due to the isoprenaline preventing general changes in lung mechanics which had previously stimulated the receptors. It is more probable that the isoprenaline prevented changes in smooth muscle con- traction local to the receptors and possibly contributing as one component 354 JANET E. MILLS AND OTHERS to the total mechanical effects. Histamine could have a direct chemical action on the end-organs, and if so then this effect might be greatly reduced by isoprenaline. With regard to the responses to embolism, anaphylaxis and injections of phenyl diguanide, we cannot say whether these interventions induced smooth muscle or mechanical changes near the receptors which thereby stimulated them, or whether theyreleased chemicals which had a histamine- like effect, direct or indirect, on the receptors. But whatever the under- lying mechanisms, ammonia, emboli, anaphylaxis and injections of hista- mine and phenyl diguanide all increased the activity ofthe irritant receptors even when ventilation was controlled during paralysis or when ventilatory changes were virtually absent because of vagotomy. The reflex action initiated from the irritant receptors has not been established directly, since no experimental procedure is known which will activate only the irritant receptors and thus provide a 'pure' stimulus. It is unlikely that the main respiratory response of the receptors is coughing, since this is not the chief change in breathing with mechanical or chemical irritation of the airways beyond the primary bronchi in experimental animals (Larsell & Burget, 1924; Widdicombe, 1954a) or probably in man (Jackson, 1922). However, there is abundant evidence that the procedures used in our experiments cause vagal reflex hyperpnoea and bronchocon- striction in a variety of mammalian species. Hyperpnoea, mediated via the vagus nerves, is produced by inhalation of ammonia and other irritants in rabbits (Cromer et al. 1933; Banister et al. 1950); by intravenous injection of histamine into rabbits (Karc- zewski & Widdicombe, 1969b), and cats and dogs (Letona et al. 1961; Nadel et al. 1966; DeKock et al. 1966); by phenyl diguanide into rabbits (Dawes et al. 1951; Karczewski & Widdicombe, 1969b); by embolism in cats, dogs and sheep (Whitteridge, 1950; Niden & Aviado, 1956; Weidner & Light, 1958; Cahill et al. 1961; Halmagyi & Colebatch, 1961); and by anaphylaxis in rabbits (Auer & Lewis, 1910; Karczewski & Widdicombe, 1969c). In the dog injection of histamine into the bronchial arterial cir- culation causes apnoea followed by hyperventilation mediated by a vagal reflex (DeKock et al. 1966). Vagally-mediated bronchoconstriction is caused by inhalation ofirritant gases in rabbits (Banister et al. 1950) and in cats and guinea-pigs (Binet & Burstein, 1946; Widdicombe, 1954a; Balchum et al. 1960; Nadel et al. 1965; Nadel et al. *1966); by intravenous injections ofphenyl diguanide into rabbits (Karczewski & Widdicombe, 1969b) and possibly cats (Barer & Niisser, 1953); by embolism (Boyer & Curry, 1944; Binet & Burstein, 1946; Niden & Aviado, 1956); and by anaphylaxis (Auer & Lewis, 1910; Karc- zewski & Widdicombe, 1969c). In the dog injection of histamine into the LUNG IRRITANT RECEPTORS 355 bronchial arterial circulation causes vagal reflex bronchoconstriction (DeKock et al. 1966). Activity in vagal efferent fibres to the trachea and lungs of cats and guinea-pigs is increased during inhalation of irritants, injection of histamine, and anaphylaxis (Vinogradova, 1955; Widdicombe, 1961a, 1966; Karczewski, 1962; and this paper), which supports the idea of a reflex component to the bronchoconstriction in these conditions. The correlation between the stimulation of lung epithelial irritant receptors by all these experimental procedures (with the exception of bronchial arterial injections ofhistamine, not tested) and vagally mediated hyperpnoea and bronchoconstriction is strong enough to point to a causal link. If this mechanism exists, the fact that both increased tidal volume and drug-induced bronchoconstriction stimulate the receptors suggests that there could be a positive feed-back tending to increase progressively ventilation and bronchomuscular tone. Some of the procedures used in our experiments are known to stimulate lung receptors other than irritant ones. Thus Paintal (1955, 1957) has shown that 'deflation receptors' in the cat can increase their discharge after embolism, and has used their consistent stimulation by phenyl diguanide as an aid to their identification. Differential block of the cervical vagus nerves by cooling to 8-10 C greatly reduces the hyperventilation and bronchoconstriction due to histamine, but has little inhibitory action on the respiratory changes due to phenyl diguanide and anaphylaxis (Karczewski & Widdicombe, 1969b, c), which suggests that more than one vagal pathway may be involved in the two latter conditions. It has not been shown how the reflexes from irritant receptors, deflation receptors and pulmonary stretch receptors interact in the complex conditions we have studied. The evidence in this paper indicates that reflexes from irritant receptors affect breathing and bronchial calibre, and therefore are a factor in the responses to the test-procedures used in this study.

We are grateful to Mr A. Husczuck for his help with some of the experiments, and to Mr F. O'Connor and Mrs M. Jolly for their efficient technical assistance. J. E. M. and H.S. were M.R.C. scholars. Part of the apparatus used was bought by grants from the Royal Society and the M.R.C.

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