Muscarinic Blockade of Methacholine Induced Airway and Parenchymal Lung Responses in Thorax: First Published As 10.1136/Thx.54.6.531 on 1 June 1999

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Thorax 1999;54:531–537 531 Muscarinic blockade of methacholine induced airway and parenchymal lung responses in Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from anaesthetised rats

M K Tulic´, J L Wale, F Peták, P D Sly

Abstract muscle. In a number of animal studies the way Background—It has previously been in which methacholine alters lung function has

shown that M1 cholinergic receptors are been shown to depend on the route of delivery. involved in the parenchymal response to Intravenous methacholine acts mainly on the inhaled methacholine in puppies using the airway producing an increase in airway resist-

M1 selective antagonist pirenzepine. Al- ance whereas inhaled methacholine alters the

though M3 receptors are responsible for mechanical properties of both the airways and acetylcholine induced bronchoconstric- the lung tissues.1–3 One possible explanation for these findings is that di erent receptors are tion in isolated rat lung, the role of M1 V receptors has not been determined in the involved. rat in vivo. Muscarinic cholinergic receptors exist in at Methods—Anaesthetised, paralysed, open least four major subtypes that can be demon- chested Brown Norway rats were me- strated pharmacologically.4 Multiple subtypes chanically ventilated and the femoral vein have, however, been identified biochemically in cannulated for intravenous injection of the lung and binding studies have shown the 56 drugs. Low frequency forced oscillations presence of M1,M2 and M3 subtypes. A spe- were applied to measure lung input im- cies diVerence in distribution of muscarinic pedance (ZL) and computerised modelling receptors of the lung has been established. In enabled separation of ZL into airway and isolated rat lung, stimulation of the M3 receptor parenchymal components. Atropine subtype is responsible for acetylcholine in- 7 (500 µg/kg iv) and pirenzepine (50, 100 or duced bronchoconstriction. However, previ- 200 µg/kg iv) were administered during ous experiments in our laboratory using alveo- steady state constriction generated by lar capsules in puppies have shown that the continuous inhalation (1 mg/ml) or intra- peripheral lung responses to inhaled metha- http://thorax.bmj.com/ venous (10 or 15 µg/kg/min) administra- choline were antagonised by the M1 selective 8 tion of methacholine. blocker pirenzepine. Results—Continuous inhalation of metha- Recent adaptations of the low frequency choline produced a 185% increase in forced oscillatory technique in rodents have enabled us to partition lung function changes frequency dependent tissue resistance (G) 1 Division of Clinical which was eVectively inhibited by atropine into airway and lung tissue components. Sciences, TVW 500 µg/kg iv (p<0.01, n = 6). Pirenzepine Peripheral lung is a complex anatomical struc- Telethon Institute for (50, 100 or 200 µg/kg) had a minimal eVect ture comprising bronchial wall (approximately Child Health Research 5%), vascular smooth muscle (8%), and alveo- on September 27, 2021 by guest. Protected copyright. & University of on the parenchymal response to inhaled 9 Western Australia methacholine. A 258% increase in airway lar ducts and walls (>86%) in rats. The Department of resistance (Raw) was induced by continu- present experiments were performed to investi- Paediatrics, Perth, ous intravenous infusion of methacholine gate whether the peripheral lung responses to Australia and and this response was eVectively abolished inhaled methacholine resulted from stimula- Department of tion of muscarinic cholinergic receptors. Based Medical Informatics by pirenzepine (p<0.001, n = 5). Cutting the vagi in the cervical region did not alter on our previous findings in puppies, the role of and Engineering, M receptors in airway and tissue responses to Albert Szent-Györgyi baseline airway mechanics. Vagotomy did 1 Medical University, not aVect lung responses to intravenous methacholine was also investigated by using the M selective antagonist pirenzepine.10 Szeged, Hungary methacholine nor the ability of piren- 1 M K Tulic´ JLWale zepine to reduce these responses. —In the rat, M -subtype re- F Peták Conclusions 1 Methods P D Sly ceptors are functional in airways but not ANIMAL PREPARATION in the tissue. Experiments were performed using adult male Correspondence to: (Thorax 1999;54:531–537) DrMKTulic´, Division of Brown Norway rats weighing 280–320 g. The Clinical Sciences, TVW rats were anaesthetised by intramuscular injec- Telethon Institute for Child Keywords: forced oscillation technique; muscarinic Health Research, P O Box blockade; lung parenchyma tion of xylazine (12 mg/kg) and ketamine 855, West Perth, WA 6872, (40 mg/kg) and placed in the supine position. Australia. The trachea was exposed in the mid cervical Methacholine is commonly used as a challenge region and cannulated with a metal catheter Received 20 July 1998 Returned to authors agent for the assessment of hyperresponsive- (internal diameter 2 mm, length 1 cm) and the 3 September 1998 ness in asthmatic subjects. It is a chemical ana- animals were mechanically ventilated (Harvard Revised manuscript received logue of acetylcholine and induces smooth Rodent Ventilator 683, MA, USA) with a tidal 24 December 1998 Accepted for publication muscle contraction by stimulating muscarinic volume of 9 ml/kg and a frequency of 1 February 1999 cholinergic receptors located on the smooth 90 breaths/minute. The femoral veins were 532 Tulic´, Wale,Peták, et al

Idaho, USA). Vagi were intact except in the third group of experiments where both right and left vagi were sectioned in the cervical Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from region before baseline measurements and Respirator administration of intravenous methacholine and pirenzepine.

ESTIMATION OF AIRWAY AND PARENCHYMAL PARAMETERS Wave tube Lung input impedance (ZL) was measured using an adaptation of the low frequency forced oscillation technique in which pressure is measured at either end of a wave tube.1 The experimental set up is shown in fig 1. A three- Loudspeaker way tap was used to switch the animal from the P1 P2 respirator to a loudspeaker-in-box system at Lateral pressure Tracheal end of expiration. The pressure in the box was at loudspeaker pressure set to 2.5 hPa to keep the transpulmonary pressure constant during measurements. The Figure 1 Schematic representation of the experimental set up. A loudspeaker is used to loudspeaker generated a small amplitude generate the low frequency forced oscillation signal which is delivered to the animal via a pseudorandom signal (15 non-integer multi- wave tube. Pressure is measured at each end of the wave tube and used to calculate the input impedance of the animal. The use of the wave tube obviated the need to measure flow ples between 0.5 and 21 Hz) through a 120 cm in small animals. During measurements the ventilator is switched out of the circuit using a long polyethylene wave tube with an internal three-way tap. diameter of 2 mm. Two identical pressure cannulated with polyethylene tubing for injec- transducers (ICS model 33NA002D) were tion of drugs. Paralysis was achieved with pan- used to measure the lateral pressures at the curonium bromide (0.2 mg/kg). The thorax loudspeaker (P1) and at the tracheal end (P2)of was opened by midline thoracotomy with the the wave tube. The P1 and P2 signals were low positive end expiratory pressure set to pass filtered (5th order Butterworth, 25 Hz corner frequency) and sampled with an ana- 2.5 cm H2O and the ribs were widely re- tracted. logue to digital board of an IBM compatible Maintenance doses of anaesthetic and mus- 1000 cle relaxant were administered intravenously every 45 minutes. The electrocardiogram and heart rate were monitored continuously using

limb leads connected to a standard electro- 750 http://thorax.bmj.com/ cardiograph (78342A, Hewlett Packard Co, O.s/l) 2 200 500 180 (cm H

160 L 140 R

O.s/l) 120 250 2 100 80 on September 27, 2021 by guest. Protected copyright. (cm H

L 60

R 0 40 20 250 0 1 10 0 –250 100

0 –750 O.s/l) –100 2 O.s/l) 2 –200 –1250 (cm H L X (cm H –300 L

X –1750 –400

–500 –2250 1 10 Frequency (Hz) 0.5 1 2 5 10 20 Frequency (Hz) Figure 2 Schematic representation of the parameters derived from the constant phase model. The input Figure 3 Real (lung resistance RL) and imaginary (lung impedance (ZL) is shown with solid lines. In the upper reactance XL) parts of lung input impedance spectra (ZL) panel the real part of ZL (resistance) can be modelled as the and the corresponding model ﬁts in a single rat after combination of a frequency independent airway resistance inhalation of saline (x) and methacholine 5 mg/ml (m). (dashed line) and a frequency dependent tissue resistance With saline, values are given as mean (SE) of 4–6 (dash-dot line). In the lower panel the imaginary part of successive ZL recordings. Following methacholine ZL (reactance) can be modelled as the combination of a administration only the peak response is reported. Open frequency independent airway inertance (dashed line) and symbols denote data points corrupted by cardiac noise and a frequency dependent tissue elastance (dash-dot line). therefore omitted from the model ﬁt. Muscarinic blockade of airway and parenchymal responses 533

120 A 0.2 B

90 /I) 2 O.s/I) O.s 2 2 60 0.1 Raw (cm H * Iaw (cm H 30

0 0 C MCh Atropine C MCh Atropine

750 C 2000 D

500 1500 ** O/I) O/I) 2 2 ** H (cm G (cm H 250 1000

0 500 C MCh Atropine C MCh Atropine

Figure 4 EVect of atropine sulphate (500 µg/kg iv) on (A) airway resistance (Raw), (B) airway inertance (Iaw), (C) http://thorax.bmj.com/ frequency dependent tissue resistance (G), and (D) tissue elastance (H) during steady state response to inhaled methacholine (1 mg/ml) in anaesthetised rats with vagi intact. Results are given as mean (SE) of six animals. *p<0.05, **p<0.01 versus methacholine response.

computer at a rate of 128 Hz. Fast Fourier omitted from the model ﬁtting since cardiac transformation with time windows of four sec- activity caused low signal-to-noise ratio at these onds and 95% overlapping was used to frequency components. A schematic represen-

calculate the pressure transfer functions (P1/P2) tation of this model is shown in ﬁg 2.

from the six second long recordings. ZL was on September 27, 2021 by guest. Protected copyright. calculated as the load impedance of the wave tube using the following equation: RESPONSES TO METHACHOLINE To standardise volume history the lungs were ZL = Zo sinh(ãL)/[(P /P )—cosh(ãL)] 1 2 hyperinflated by superimposing two inspira- where L is the length, Zo is the characteristic tory cycles before starting measurements. Fol- impedance, and is the complex propagation ã lowing 15 minutes of mechanical ventilation, wave number of the wave tube. The latter two baseline respiratory mechanics were measured parameters are determined by the geometrical by recording 4–6 ZL spectra. These were data and the material constants of the tube wall ensemble averaged and the model fitted to the and the air. average spectrum. Methacholine was then con- To separate the airway and parenchymal tinuously administered in a concentration suf- mechanics, a model containing a frequency ficient to increase frequency dependent tissue independent airway resistance (Raw) and resistance (G) by 50–100% at the steady state inertance (Iaw) in series with a constant phase response. tissue model including frequency dependent tissue resistance (G) and tissue elastance (H) was fitted to the ZL spectra by minimising the INHALED METHACHOLINE diVerences between the measured and mod- Methacholine was administered to the animal elled impedance values using the following by inhalation at a concentration of 1 mg/ml equation: using a jet nebuliser (LC PLUS, Pari-Werk ZL =Raw+jùIaw + (G—jH)/ùá GmbH, Germany) driven by compressed air where j is the imaginary unit, ù is the angular (5 l/min) into the inspiratory port of the respi- frequency (2ðf), and á, which is expressed as á rator. This nebuliser delivers particles approxi- =2/ðarctan (H/G), which is not an independ- mately 5 µm in size (manufacturer’s specifica- ent parameter. Impedance points coinciding tions). Steady state constriction was obvious with the heart rate and its harmonics were after 8–12 minutes. 534 Tulic´, Wale,Peták, et al

120 A 0.2 B Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from

90 /I) 2 O.s/I) O.s 2 2 60 0.1 Raw (cm H Iaw (cm H 30

0 0 C MCh PZ50 PZ100 C MCh PZ50 PZ100

750 C 2000 D

500 1500 O/I) O/I) 2 2 H (cm G (cm H 250 1000

0 500 C MCh PZ50 PZ100 C MCh PZ50 PZ100

Figure 5 EVect of pirenzepine 50 µg/kg iv (PZ50) or 100 µg/kg iv (PZ100) on (A) airway resistance (Raw), (B) http://thorax.bmj.com/ airway inertance (Iaw), (C) frequency dependent tissue resistance (G), and (D) tissue elastance (H) during steady state response to inhaled methacholine (1 mg/ml) in anaesthetised rats with vagi intact. Results are given as mean (SE) of ﬁve animals.

INTRAVENOUS METHACHOLINE tion induced by intravenous methacholine was Methacholine was administered intravenously investigated following baseline measurements. by continuous infusion at a rate of 10–15 µg/kg/ The infused concentration of methacholine min (Stoelting 220VAC, USA), suYcient to used to approximately double Raw was 10 µg/ increase airway resistance (Raw) by approxi- kg/min in four rats and 15 µg/kg/min in one on September 27, 2021 by guest. Protected copyright. mately 100%. Individual ZL curves were animal. collected every two minutes until a plateau response was observed (approximately 15 minutes). These procedures resulted in a total MATERIALS methacholine administration time of 25–35 Pirenzepine was kindly donated by Boehringer minutes. Mannheim GmbH (Mannheim, Germany); atropine sulphate and pancuronium bromide RESPONSES TO MUSCARINIC ANTAGONIST was purchased from Astra Pharmaceuticals After the steady state response to methacholine (North Ryde, NSW, Australia); acetyl-â- had been established, the antagonist was methylcholine chloride (methacholine) was administered by intravenous injection. After a obtained from Sigma Chemical Company (St period of two minutes individual ZL curves Louis, MO, USA); ketamine was purchased were collected also at two minute intervals. from Troy Laboratories (Smithﬁeld, NSW, Atropine sulphate (500 µg/kg) was adminis- Australia); xylazine from Bayder (Pymble, tered intravenously during the steady state NSW, Australia); and pentobarbitone sodium response to inhaled methacholine. Pirenzepine from Virbac (Peakhurst, NSW, Australia). was administered in cumulative doses of 50, 100 or 200 µg/kg at approximately 10–15 minute intervals during steady state response STATISTICAL ANALYSIS to either inhaled or intravenous methacholine. Comparisons between groups were made on log transformed data using one way analysis of EFFECTS OF CERVICAL VAGOTOMY variance (ANOVA) with Student-Newman- In animals with the vagi cut, the eVect of Keuls correction for multiple comparisons. All cumulative administration of pirenzepine (50, results are expressed as mean (SE). p values of 100, or 200 µg/kg) during steady state constric- <0.05 were regarded as statistically signiﬁcant. Muscarinic blockade of airway and parenchymal responses 535

120 A 0.2 B Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from

90 /I) 2 O.s/I) O.s 2 2 60 0.1 Raw (cm H Iaw (cm H 30

0 0 C MCh PZ50 PZ100 C MCh PZ50 PZ100

750 C 2000 D

500 1500 O/I) O/I) 2 2 H (cm G (cm H 250 1000

0 500 C MCh PZ50 PZ100 C MCh PZ50 PZ100

Figure 6 EVect of pirenzepine 50 µg/kg iv (PZ50) or 100 µg/kg iv (PZ100) on (A) airway resistance (Raw), http://thorax.bmj.com/ (B) airway inertance (Iaw), (C) frequency dependent tissue resistance (G), and (D) tissue elastance (H) during steady state response to intravenous methacholine (10 mg/kg/min) in anaesthetised rats with vagi intact (C,n=5)orvagicut (x, n = 5). Results are given as mean (SE). Results EFFECT OF PIRENZEPINE ON INHALED LUNG FUNCTION IN RESPONSE TO INHALED METHACHOLINE METHACHOLINE During steady state constriction induced by Impedance spectra at baseline and after metha- inhaled methacholine G was increased by choline challenge are shown in fig 3. During 153% above baseline (p<0.01, n = 5; fig 5) and steady state constriction induced by continuous was associated with a corresponding 132% on September 27, 2021 by guest. Protected copyright. inhalation of methacholine, frequency depend- increase in tissue elastance (p<0.05, n = 5). ent tissue resistance (G) was increased by 185% Once again, no change in Raw or Iaw was evi- above baseline (p<0.01, n = 6; fig 4). This dent with inhaled methacholine. Pirenzepine increase in G was associated with a similar (50 µg/kg iv) produced no eVect on parenchy- 142% increase in tissue elastance (H) (p<0.01, mal responses to methacholine (fig 5). Increas- n = 6). Inhaled methacholine produced only ing the cumulative dose of pirenzepine to minimal changes in airway resistance (Raw) and no change in airway inertance (Iaw) (fig 4). 100 µg/kg (fig 5) and 200 µg/kg (results not shown) produced no additional eVect. EFFECT OF ATROPINE ON INHALED METHACHOLINE LUNG FUNCTION IN RESPONSE TO INTRAVENOUS Following intravenous injection of atropine METHACHOLINE: VAGI INTACT (500 µg/kg iv) parenchymal responses to inhaled The airway and tissue steady state responses methacholine were inhibited, significantly re- to continuous intravenous infusion of ducing G by 62% (p<0.01, n = 6) and H by 77% (p<0.01, n = 6; fig 4). While G was still signifi- mthacholine are shown in fig 6. Intravenous cantly above the baseline or control value after methacholine induced a marked and statis- atropine treatment (p<0.001), tissue elastance tically significant increase in airway resistance (H) returned to the baseline level after atropine of 258% above the baseline value (p<0.001, administration. Despite the lack of eVect of n = 6; fig 6A) and an increase of 165% methacholine on Raw, intravenous injection of in frequency dependent tissue resistance atropine significantly reduced the baseline air- (p<0.01, n = 6; fig 6C). Intravenous adminis- way tone by 20% (p<0.05, n = 6). Little change tration of methacholine produced no eVect on was observed in Iaw with methacholine or atro- airway inertance (fig 6B) or tissue elastance pine administration. (fig 6D). 536 Tulic´, Wale,Peták, et al

EFFECT OF PIRENZEPINE ON INTRAVENOUS Discussion METHACHOLINE: VAGI INTACT The results of these experiments in anaesthe- Intravenous injection of pirenzepine (50 µg/ tised Brown Norway rats show that prolonged Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from kg) resulted in a rapid decline in both the air- inhalation of methacholine results in a pre- way and tissue response to methacholine, dominantly parenchymal (or tissue) constrictor reducing Raw by 76% (p<0.001, n = 6) and G response which is eVectively reversed by a by 73% (p<0.001, n = 6) from the plateau maximum dose of atropine sulphate but not by steady state methacholine response (fig 6). Iaw pirenzepine. In contrast, intravenous metha- was significantly reduced by 21% below base- choline produced more marked changes in air- line (p<0.05, n = 6) and H remained way resistance which were reversed by piren- unchanged (fig 6). The maximum eVect on zepine. Raw and G was evident within five minutes Our adaptation of the low frequency forced after administration and was maintained over oscillation technique allows lung mechanics to the period of 15 minutes in which the response be partitioned into airway and parenchymal was observed. components by fitting the constant phase Increasing the dose of pirenzepine to model to the impedance spectra. The airways 100 µg/kg resulted in further reduction in air- through which gas moves by bulk flow will be way resistance to 97% from plateau metha- included in the airway component, whereas choline response or 3% above baseline airways through which gas moves by diVusion (p<0.05, n = 6), completely abolishing the air- are likely to be included in the parenchymal way response to methacholine. Similarly, G component. It is therefore most likely that the was further reduced to 77% of the metha- parenchymal response is the result of contrac- choline plateau response (p<0.001, n = 6; fig tion of smooth muscle in respiratory bronchi- 6). The response in G was still significantly oles and alveolar ducts and of contractile higher than the baseline response (p<0.05). elements in the alveolar septa. These may be Increasing the pirenzepine dose to 200 µg/kg the contractile elements described by Kapanci iv (results not shown) did not result in further et al.11 Vascular smooth muscle is present in the attenuation of the parenchymal responses to lung parenchyma but may be expected to relax intravenous methacholine. in response to methacholine.12 Both atropine and cervical vagotomy tended to decrease Raw. Due to the variability, this reached statistical LUNG FUNCTION IN RESPONSE TO INTRAVENOUS significance only for atropine. The lack of effect METHACHOLINE: VAGI CUT on baseline parenchymal measurements is Cervical vagotomy produced no eVect on consistent with cholinergic innervation being baseline lung function parameters when com- most dense in the large airways.13 Methacholine pared with animals with vagi intact (fig 6). In

responses on airways were not altered by http://thorax.bmj.com/ the airway compartment a small reduction in removing vagal tone. the baseline Raw (15%) in the rats with vagi cut Atropine is a non-selective muscarinic an- failed to reach statistical significance. With the tagonist with very similar potency on all vagi cut, the airway response to intravenous muscarinic receptor subtypes.14 It was eVective methacholine was similar to that seen with the in the present experiments in reducing the vagi intact (fig 6), with Raw increasing by parenchymal responses to inhaled metha- 284% (p<0.05, n = 5) above baseline. The choline, confirming the role of muscarinic same pattern of response was also seen for G receptors in producing this response. A signifi- which increased by 191% from baseline cant reduction in Raw was also apparent. on September 27, 2021 by guest. Protected copyright. (p<0.01, n = 5). In rats with cervical vagotomy Pirenzepine demonstrates relative selectivity only, minor but significant increases in Iaw for M subtypes.14 15 In the present model (12%) and H (16%) were apparent after 1 pirenzepine inhibited the airway responses to methacholine administration (fig 6). intravenously administered methacholine but, unlike atropine, did not reduce the tissue EFFECT OF PIRENZEPINE ON INTRAVENOUS responses to inhaled methacholine. The lowest METHACHOLINE: VAGI CUT dose of pirenzepine (50 µg/kg iv) was approxi-

Administration of pirenzepine in a dose of mately the ED50 dose, as reported by other 50 µg/kg iv eVectively reduced both the airway workers measuring inhibition of the increased

and the parenchymal response to intravenous blood pressure with the M1 selective agonist methacholine, reducing Raw by 78% (p<0.05, McN-A-343 in anaesthetised rats.15 In the n = 5) and G by 67% (p<0.05, n = 5) from the present experiments pirenzepine reduced the plateau steady state methacholine response. airway response to intravenously infused Pirenzepine in a dose of 50 µg/kg iv signifi- methacholine by 76% and 78% in rats with cantly reduced the baseline response in Iaw vagi intact and severed, respectively. Piren- (p<0.01, n = 5) and H remained unchanged zepine (100 µg/kg iv) had an almost maximal (fig 6). Increasing the dose of pirenzepine to eVect on the Raw to intravenous methacholine 100 µg/kg further reduced Raw to 94% of the and yet, at this dose, there was no significant plateau response (p<0.001, n = 5) with no reduction in tissue responses to inhaled metha- additional significant eVect on G (fig 6). Piren- choline. It is therefore likely that pirenzepine is

zepine 200 µg/kg (results not shown) did not not antagonising M3 receptors at these concen- produce any further eVect. The pattern and trations. As pirenzepine has a selectivity proﬁle 14 15 time course of the pirenzepine response in ani- of M1 >M4,M3 >M2, it is also unlikely that

mals with vagi cut was similar to the responses it is acting on M2 receptors and one must con-

in animals with vagi intact. clude that we are observing an M1 eVect on Muscarinic blockade of airway and parenchymal responses 537

Raw. Few M1 subtype muscarinic receptors increase in parenchymal resistance produced have been identiﬁed in rat lung using binding by prolonged inhalation of methacholine. The

56 Thorax: first published as 10.1136/thx.54.6.531 on 1 June 1999. Downloaded from studies, and only M2 and M3 receptors were M1 selective muscarinic antagonist pirenzepine evident in the trachea and bronchi.6 Up to 91% had no signiﬁcant eVect on parenchymal of muscarinic receptor protein in rat lung is of responses to inhaled methacholine and re-

the M2 subtype with M3 receptor protein mak- versed airway responses to intravenously ad- ing up most of the remainder.8 The apparently ministered methacholine. Cutting the cervical

small number of M3 receptors has, however, vagi did not alter resting lung function or been reported to dominate muscarinic responses to methacholine and pirenzepine. It 7 responses. It is therefore possible that a small therefore appears that, in the rat, M1 subtype

number of M1 receptors will still be suYcient to receptors are functionally present in the produce a physiological response. airways but could not be demonstrated in the Pirenzepine reversed the changes in both parenchyma. Raw and G when constriction was induced by intravenous methacholine. The increase in G This study was supported by a grant from the National Health with methacholine was not, however, associ- and Medical Research Council, Australia and an Australian ated with an increase in H. Lutchen et al16 Lung Foundation, Smith Kline and Beecham Award. M K Tulic´ is holding an Asthma Foundation of Western Australia investigated this phenomenon using ventilation Postgraduate PhD Scholarship. with gas mixtures of diVering viscosities and densities. They explained the increase in G as 1 Peták F, Hantos Z, Adamicza Á, et al. Methacholine-induced resulting from heterogeneous airway constric- bronchoconstriction in rats: eVects of intravenous vs aero- tion. With this background we can interpret sol delivery. J Appl Physiol 1997;82:1479–87. our results as follows. Pirenzepine competi- 2 Sly PD, Lanteri CJ. DiVerential responses of the airways and pulmonary tissues to inhaled histamine in young dogs. J tively reverses the highly inhomogeneous con- Appl Physiol 1993;68:1562–7. striction of the bronchial tree present following 3 Sly PD, Lanteri CJ. Partitioning of pulmonary responses to inhaled methacholine in puppies. J Appl Physiol 1991;71: intravenous methacholine, therefore decreas- 886–91. ing the inhomogeneities present in the periph- 4 Eglen RM, Hegde SS, Watson W. Muscarinic receptor subtypes and smooth muscle function. Pharmacol Rev ery and producing a decrease in G. Taking into 1996;48:531–65. account the pirenzepine results when lung 5 Gies JP, Bertrand C, Vanderheyden P, et al. Characterization of muscarinic receptors in human, guinea pig and rat lung. constrictor tone is induced either by aero- J Pharmacol Exp Ther 1989;250:309–15. solised or intravenously administered metha- 6 Fryer AD, El Fakahany EE. Identiﬁcation of three muscarinic receptor subtypes in rat lung using binding choline, our data suggest that pirenzepine acts studies with selective antagonists. Life Sci 1990;47:611–8. only on the airways since a decrease in G was 7 Post MJ, Te Biesebeek JD, Doods HN, et al. Functional characterization of the muscarinic receptor in rat lungs. Eur not evident when a purely parenchymal J Pharmacol 1991;202:67–72. response to inhaled methacholine was gener- 8 Sly PD, Willet KE, Kano S, et al. Pirenzepine blunts the pulmonary parenchymal response to inhaled methacholine.

ated. Pulm Pharmacol 1995;8:123–9. http://thorax.bmj.com/ M receptors are understood to be associated 9 Ludwig MS, Dallaire MJ. Structural composition of lung 1 parenchymal strip and mechanical behaviour during with neuronal tissue to facilitate parasympa- sinusoidal oscillation. J Appl Physiol 1994;77:2029–35. 17 thetic ganglionic transmission which, with the 10 Hammer R, Giachetti A. Muscarinic receptor subtypes: M1 and M2. Biochemical and functional characterization. Life generalisation that autonomic innervation de- Sci 1982;31:2991–8. creases down the respiratory tree,13 would 11 Kapanci Y, Assimacopoulos A, Irle C, et al. Contractile interstitial cells in pulmonary alveolar septa: a possible agree with the purely airway eVects observed in regulator of ventilation/perfusion ratio? Ultrastructural our experiments. They were also present in immunofluorescence and in vitro studies. J Cell Biol 1974; 60:375–92. human alveolar walls in human lungs but not in 12 Barnes PJ. Modulation of neurotransmission in airways. 18 the guinea pig. The ability of pirenzepine to Physiol Rev 1992;72:699–729. on September 27, 2021 by guest. Protected copyright. 13 Richardson JB. Nerve supply to the lungs. Am Rev Respir Dis reverse the airway responses to methacholine 1979;119:785–802. could be explained if methacholine resulted in 14 Doods HN, Entzeroth M, Ziegler H, et al. Pharmacological profile of selective muscarinic receptor antagonists on a positive feedback, via ganglionic M1 recep- guinea-pig ileal smooth muscle. Eur J Pharmacol 1994;253: tors, resulting in increased release of acetylcho- 275–81. 15 Eberlein WG, Engel W, Mihm G, et al. Structure-activity line from cholinergic nerves. However, the fact relationships and pharmacological profile of selective tricy- that the same pattern of response was seen after clic antimuscarinics. Trends Pharmacol Sci 1989;Suppl IV (Subtypes of muscarinic receptors):50–4. bilateral cervical vagotomy makes this explana- 16 Lutchen KR, Hantos Z, Peták F, et al. Airway inhomogenei- tion unlikely. While caution must be exercised ties contribute to apparent lung tissue mechanics during constriction. J Appl Physiol 1996;80:1841–9. when extrapolating from the results of animal 17 Barnes PJ. Muscarinic receptor subtypes in airways. Life Sci studies to humans, clinical studies have shown 1993;52:521–7. 18 Mak JCW, Barnes PJ. Autoradiographic visualization of that pirenzepine improves lung function in muscarinic receptor subtypes in human and guinea pig both healthy subjects19 and asthmatics.20 lung. Am Rev Respir Dis 1990;141:1559–68. 19 Cazzola M, Russo S, DeSantis D, et al. Respiratory In conclusion, the present experiments in responses to pirenzepine in healthy subjects. Int J Clin anaesthetised rats show that, using lung Pharmacol 1987;25:105–9. impedance measurements, the non-selective 20 Sertl K, Meryn S, Graninger W, et al. Acute eVects of pirenzepine on bronchospasm. Int J Clin Pharmacol Therapy muscarinic antagonist atropine reverses the Toxicol 1986;24:655–7.