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 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 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.