Methods of study of airway smooth muscle and its physiology

Pharmac. Ther. Vol. 7, pp. 253-295. © Pergamon Press Ltd. Printed in Great Britain.

0163-7258/79/1101 0253/$5.00/0

Specialist Subject Editor: J. G. WIDDICOME

METHODS OF STUDY OF AIRWAY SMOOTH MUSCLE AND ITS PHYSIOLOGY H. L. H A H N Medizinische Universitdts-Poliklinik, Klinikstrasse 8, 8700 14rdrzburg, West Germany

and J. A. NADEL Cardiovascular Research Institute, University of California Medical Center, San Francisco, CA 94143, USA

The emphasis of this review is on methods. Important physiological findings will be mentioned in the context of the method used in their discovery. These findings and possible functions of airway smooth muscle will be briefly summarized at the end. 1. METHODS OF STUDYING AIRWAY SMOOTH MUSCLE I N VIVO In vivo we cannot measure smooth muscle tone directly, but must infer changes in tone from measurements reflecting airway calibre. These can be either functional (e.g. airway resistance, maximal flow) or morphological (e.g. bronchography). 1.1. MEASUREMENTS OF RESISTANCE

The resistance of the airways is defined, in analogy to Ohm's Law, as the difference in pressure between aiveoli (Pair) and airway opening (Poo), divided by the flow (12) caused by this pressure difference: Raw =Posv - Poo/f/(dimensions: cm H20/i. sec. Fig. 1). If the difference between pleural pressure (Pp~) and mouth pressure (Poo) is related to flow, the resistance obtained is that of airways (Row)plus lung tissue (Ra), called pulmonary or lung resistance, (RL): Rr = Row + Ra = Ppt - P.o/f~. Finally, if mouth pressure is related to ambient pressure (Pot,.), the resistance to airflow offered by the chest wall (Row) is also included and one obtains the resistance of the whole respiratory system (R.): R,s = R°. + Ra + Rc~ = Poo - Po,.J(/.

Patm

~o

I I I I I I.

i I I ,

,

Raw

RE Rrs

FIG. 1. Resistance of airways (R°w),lung (RL) and respiratory system (R,,). The circle represents the aiveoli emptying via the airways to the mouth, the box represents the thorax, and the pressure inside it is pleural pressure (Pp~). The difference between alveolar (Pate) and mouth pressure (P°o) is the driving pressure for flow. 253

254

H.L. HAHN and J. A. NADEL

/

o

c: o

.2 he"

i

RV

I

I

I

!

TLC

FRC Volume

RV

FRC Votume

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FIG. 2. Relationship of airway resistance (top) and conductance (bottom) to lung volume. Since the conductancevolume plot does not go through the origin, 'specific' conductance (conductance/lung volume) is not entirely independent of lung volume.

Pharynx

Raw(cm H20/1. sec)

Bronchi

0,2

<2ram

Total : 1,2

FIG. 3. Longitudinal distribution of airway resistance in a normal person. The narrowing of individual airways is more than made up by a large increase in the number of airways, so total cross-sectional area increases greatly towards the periphery.

~o,,~

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A Pbox

& (I A Pbox

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~ &PQo=Af

~

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~

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FIG. 4. Measurement of airway resistance and thoracic gas volume (TGV) by plethysmography. As the subject pants back and forth into the box (left), bulk movement of air does not change box pressure. Changes in box pressure (APbox) are due to the changes in alveolar pressure (compression, rarefaction) causing gas flow (AI;'). AI?/AP~. is slope (1). As the subject pants against the closed shutter (right), pressure equilibration occurs throughout the lung because there is no flow, so mouth pressure (P=o)equals alveolar pressure (Po~v)and can be related to changes in box pressure (AP~o,), giving slope (2). Division of slope (2) by slope (1) gives airway resistance. TGV is measured from procedure 2 by applying Boyle's law : (TGV x P) = (TGV + AV) × (P - Ap). Since we know P (barometric minus water vapour pressure), Ap (from Poo, when shutter is closed) and AV ( = change in TGV, from APbo, which has previously been calibrated against a known pump volume--not shown here), we can solve for the unknown TGV.

Methods of study of airway smooth muscleand its physiology

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During laminar flow, the resistance of a single tube is given by Poiseuille's equation : R = 8Lp/rrr 4, where L = length and r = radius of the tube and tt = gas viscosity. Obviously tube radius (lumen width) is the major determinant of resistance (R • 1/r 4) and this remains true although the application of the PoiseuiUe equation to the airways is a simplification. Changes in resistance are assumed to be due to changes in smooth muscle tone (and not, for example, mucosal swelling or mucus secretion) if: (1) they are rapid, (2) they can be reversed by fl-adrenergic agonists, which relax airway smooth muscle, (3) post mortem examination of the tissue can exclude other causes (e.g. Nadel et al., 1964). Of the many variables affecting airway resistance - phase of respiration (Bouhuys and Jonson, 1967; Froeb and Mead, 1968; Vincent et al., 1970), volume history (Nadel and Tierney, 1961), flow rate (Bouhuys and Jonson, 1967) and lung volume (Briscoe and DuBois, 1958) - lung volume is the most important and has to be considered both in comparing resistance values in individuals of differing size (adults vs children) and in the same individual on different occasions. Because of the curvilinear relationship between resistance and lung volume (Fig. 2) it is more convenient to use the reciprocal of resistance, i.e. conductance (Gow, Gt., G,,, in l./sec cm H20), which is then divided by the absolute lung volume at which it is measured to give specific conductance SG°w (SGL, SG,,). Specific resistance (SRow) equals resistance times the volume at which it is measured. In healthy subjects, most of the resistance is located in the central intra- and extra-thoracic airways, while peripheral airways of less than 2 mm diameter contribute little resistance (Fig. 3). The resistance of the larynx can comprise close to 50 per cent of Raw and varies markedly during different respiratory manoeuvres (Hyatt and Wilcox, 1961 ; Ferris et al., 1964). Techniques used to measure resistance:

1. 1. 1. Body plethysmography The principle is based on Boyle's law and has long been known (e.g. Sonne, 1923). The method measures Potv and this is used to obtain airway resistance (Row) in a simple, noninvasive procedure (Fig. 4). That changes in P,~ during panting are isothermal (a condition for applying Boyle's law) was confirmed by Mead and Collier (1959) and Nolte (1969). In addition to measuring Raw, plethysmographic methods have the added advantage of measuring the thoracic gas volume (TGV) at which the manoeuvre was performed. Three types of plethysmographs exist. 1.1.1.1. The variable pressure plethysmooraph ('pressure box', Fig. 4). Pressure changes in the plethysmograph are measured by a technique developed for practical use by DuBois et al. (1956a) described in detail by Comroe et al. (1959) and is currently the most widely used type of plethysmograph. Some technical points have to be observed. When the subject pants in and out there is a small additional artefact due to the change in box pressure (APaox) because of differences in temperature and humidity of the gas exchanged. This can be prevented by (a) shallow panting, which keeps most of the gas inside the heated pneumotachograph (DuBois et al., 1956a), (b) re-breathing gas at BTPS conditions from a bag attached to the other side of the pneumotachograph (Jaeger and Otis, 1964; Sackner et al., 1964; Jonson and Bouhuys, 1967) or making the whole box BTPS (Bartlett et al., 1959) and (c) electronic correction of the signals (Muysers et al., 1969 ; Smidt et al., 1969). Shallow panting has two other advantages. Firstly, it reflexly maintains the glottis open, reducing its contribution to total Raw and also reducing the variability of the measurement (Stanescu et al., 1972b; Barter and Campbell, 1973). This manoeuvre can also be used in other measurements of resistance, e.g. by forced oscillation (Vincent et al., 1970). Secondly, it improves the signal-to-noise ratio by making the flow rate signal larger and the volume exchanged smaller than during tidal breathing. To minimise thermal drift of Pb°x, Lloyd and Wright (1963) installed a small leak in the box wall which allowed slow thermal changes to occur without distorting rapid oscillations such as occur with panting. To dampen acoustic noise (another problem because of high transducer sensitivity) the reference end of the pressure transducer can be connected to large

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compensating chambers (Comroe et al., 1959). Bryant and Hansen (1975) rediscovered a device used earlier by Bartlett et al. (1959) where the reference end of the transducer was connected through a Y-piece to a 25-gauge needle and to a compensating vessel inside the box. The pneumatic resistance of the needle allowed thermally induced pressure changes but not fast oscillations to pass, the compensating vessel dampened acoustic noise and maintained transducer sensitivity. 1.1.1.2. Volume displacement plethysmograph (Mead, 1960b; Sonne, 1923). Volume changes are measured directly with a spirometer attached to the box wall, not indirectly through changes in Pbox(substitute AVbo:, for AP~o~ in Fig. 4). If the mouth-piece is connected through a hole in the box wall to the outside, the spirometer records changes in TGV due to exchange of bulk volume (in addition to those caused by gas compression, which a spirometer attached to the mouth will not measure). This plethysmograph is suitable for recording large volume events, but its lower frequency response causes problems in recording small volume changes during rapid events. The 'pressure compensated volume plethysmograph' was a considerable improvement: since any lag in the response implied transient pressure changes in box and spirometer, addition of this pressure signal, adequately amplified, to the volume signal gave the initial volume displacement (Grimby et al., 1968 ; Clement and van de Woestijne, 1969) and the response could be further improved by proper damping (van de Woestijne and Bouhuys, 1969). Temperature control inside the box is critical and is usually achieved by air conditioning (Grimby et al., 1968; Jonson and Bouhuys, 1967). The pressure box is usually used for fast events (e,g. measurement of Ra~), whereas the volume box is superior for slower events and to record large volume changes. In many studies both types are used in combination. 1.1.1.3. Theflow plethysmograph. In this box the spirometer is replaced by a pneumotachograph in the box wall (Jonson, 1969; Stanescu et al., 1972a). Volume changes are obtained by integration of the flow signal. Thermostatic temperature control and pressure compensation are needed as for the volume box (Stanescu et al., 1972a; Finucane et al., 1975). The latter is particularly simple because the same transducer can be used to measure flow and P~ax, the reference end of this very sensitive instrument being again connected to a compensating chamber (Jonson, 1969 ; Finucane et al., 1975). With pressure correction, frequency response is superior to that of the spirometer volume box (35 c/see in Jonson, 1969) and the flow box is widely used for human and animal studies (e.g. Drazen et al., 1976a). It combines advantages of the pressure (extended frequency response) and volume boxes (smaller size, less severe requirements for air tightness, possibility of recording slow and larger volume changes), but attention has to be paid to problems of integrator drift and alinearity of the flow meter. 1.1.1.4. Plethysmography in babies, anaesthetized subjects and animals. For measurement of R°w during tidal breathing babies (Radford, 1974; Stocks et al., 1977) or animals (Smejkal and Pale~ek, 1972) rebreathe warm moist air from a bag to prevent thermal exchange, bearing in mind that hypoxaemia and hypercapnia may affect bronchomotor tone (Nadel and Widdicombe, 1962b). Measurement of TGV is made possible by the fact that breathing movements continue against the closed shutter (Mead and Collier, 1959; Wohl et al., 1968 ; Robinson and Gillespie, 1973; Koo et al., 1976). Row and TGV can also be measured by panting manoeuvres: Wanner and Sackner (1973) produced panting in dogs by transvenous electrical stimulation of the phrenic nerve and Avery and Sackner (1972) by using a special compression apparatus. A double chamber method developed to measure Raw in small animals made use of the fact that during flow the difference between the volume measured at the mouth and TGV is related to Patv (Johanson and Pierce, 1971). In paralyzed animals the method of Nisell and DuBois (1954) can be used to measure TGV: injection of 10-20 ml of air into the animal behind the closed shutter (AV in Boyle's Law) is followed by rapid pressurization of the plethysmograph (AP) until transthoracic pressure is zero as before injection. From AV, AP and the initial Pa~v(Pat,, minus water vapour pressure) the unknown TGV is calculated. The method was used again by Laver et al. (1964) and was improved by

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Colebatch and Engel (1974a, b). Potvhas also been measured directly with needles (Hoppin et al., 1978) and with button-like or hemispherical lucite capsules glued to the lung surface, underneath which the pleura was punctured several times while the dome of the capsule was connected to a pressure transducer (e.g. Sasaki et ai., 1977); this can only be done in open chest animals or in excised lungs. The devices themselves can undoubtedly change Patv by altering local stresses and strains but in practice the error has been found to be small. 1.1.2. Subtraction methods These methods are commonly used to measure RL in animals. Transpulmonary pressure (Prp, the difference between mouth and pleural pressure) is obtained, the static elastic component is subtracted from it and the remaining resistive component is divided by flow to obtain RL. None of the methods provides a simultaneous measurement of TGV, which has to be obtained separately, e.g. by dilution methods. 1.1.2.1. Measurement of transpuimonary pressure (PTP). PrP can be measured in one of three ways: as difference between mouth and pleural pressure (Pp~), as difference between mouth and oesophageal pressure (Poes) or as mouth pressure (Pao) in open chest animals when Pp~ is zero. P~ is obtained most simply from a needle in the pleural space (yon Neergaard and Wirz, 1927a; Mead and Whittenberger, 1953; Diamond, 1967), but a needle is only suitable for short periods of recording because it tends to become obstructed. Special rigid catheters, usually with side holes (Farhi et ai., 1957; Giles et ai., 1971) and flat button-like Starling resistors (McMahon et al., 1969) have been used but the most commonly used devices are air filled balloons, flexible catheters of the mushroom (Mead and Collier, 1959; Wohl et al., 1968) or malecot type (Nadel et al., 1964; Colebatch and Engel, 1974b) or a continuous piece of tubing with several holes in the middle pulled through the pleural space of small animals (Amdur and Mead, 1958). A small pneumothorax is often created because it dampens unwanted oscillations produced by the heart beat, keeps the catheter from becoming obstructed and has been shown not to affect the accuracy of the measurements (Wohl et al., 1968; Hoppin et al., 1969). A button-like double-disc device with openings in the stem connecting the discs for chronic recordings of Po~ in conscious dogs has been described by Tosev et al. (1969). In animals in the supine position, insignificant pressure gradients are measured between top and bottom of the lung (Farhi et al., 1957; McMahon et al., 1969; Hoppin et ai., 1969). Oesophageal pressure (P~,) is usually measured with a balloon tipped catheter placed in the middle or lower third of the oesophagus. The balloon contains a small volume of air (0.4-0.6 ml) to prevent obstruction of the several openings near the end of the catheter. Pressure-volume characteristics of the balloon influence the measurement (Milic-Emili et al., 1964; Lemen et al., 1974) and for certain applications it may become necessary to measure ,toes at more than one balloon volume and extrapolate back to zero volume (Gillespie et al., 1973). This is unnecessary when only changes in Poe, have to be measured. Fry et al. (1952) found Put to be slightly more negative than Po,~ in man and this has been confirmed in dogs especially when supine (Mead and Whittenberger, 1953; Wohl et al., 1968; Gillespie et al., 1973; Gillespie and Hyatt, 1974). Despite these differences in absolute values, changes in Po~ have been shown to be reflected accurately. The technique most commonly used in dogs (animal prone, simultaneous introduction of a suction catheter to keep the oesophagus emptied of air and liquid etc.) has been described by Wohl et al. (1968). Helium-filled catheters have a better frequency response than air-tiMed catheters (Fry et al., 1952). To open the chest most investigators use a sternal splitting operation. An alternative is to open one of the intercostal spaces with a wide cut and to keep it open with a retractor. The expiratory line of the ventilator must be connected to an underwater seal to prevent collapse of the lung, but the bubbling makes the P r e trace rather noisy. Parker et al. (1963) have described a special pump that maintains a controlled and constant tidal volume, withdrawing exactly the volume inspired; a potentiometer and generator attached to the pump piston generate signals for volume and flow rates. Another way to measure R~ in

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animals with open chests is by forced oscillations with the ventilator turned off temporarily. 1.1.2.2. Determination of compliance (CL). At normal breathing frequencies, Pre has two components: one needed to overcome elastic recoil (elastic component, Pe~)which is present even under static (no flow) conditions, and one needed to overcome the resistance to air flow (resistive component) which is present only during flow. The elastic properties of the lungs are expressed as the distensibility or compliance of the lung and are measured as the change in lung volume (l. or ml) produced by a change of 1 cm H 2 0 in static distending pressure (Pet) across the lung (l./cm H 2 0 or ml/cm H20). To measure static lung compliance, the subject inhales to full inflation and then exhales slowly, change in volume being measured in a volume plethysmograph or as expired volume. At intervals the mouthpiece is occluded and Pre is measured in the absence of flow. Static PTP = Pe~ is plotted against the simultaneously measured lung volume to obtain the static pressure-volume (PV) curve, the slope of which (AV/AP = compliance, CL) is approximately linear in the tidal volume range. RL at each lung volume can be obtained by repeating the manoeuvre dynamically, i.e. without the breathholding stops, and by dividing the flow measured at each lung volume into the difference between static and dynamic PrP (Frank et al., 1957). More often, dynamic CL is measured ; this is the compliance during normal breathing. At the beginning and end of inspiration, no air flows so the volume change between these two points of zero flow divided by the accompanying PrP change is dynamic compliance. 1.1.2.3. Subtraction procedures to separate R L and CL. All of these assume linearity of CL in the tidal volume range; most can also be used to separate R,, from C , (= compliance of the whole respiratory system), when Pao is related to Patm (Comroe et al., 1954; Bergman and Waltemath, 1974). (a) Method of yon Neergaard and Wirz (1927b, Fig. 5). A straight line is drawn connecting the two pressures (Pre) obtained at zero flow points. The difference between this line and total Pre represents the resistive portion of Pre. Divided by flow it gives instantaneous RL at any point in the respiratory cycle. (b) Isovolume technique (Fig. 6). PrP is measured at the same volume during inspiration and expiration. Since at the same volume Pet is the same, differences in Pre between isovolume points are related to differences in flow, division of APre by AI? gives RL. The method gives the average RL over the tidal volume range and has been used frequently (e.g. Frank et al., 1957; Admur and Mead, 1958; Diamond, 1967). Several computer versions have been developed (Giles et al., 1971 ; Battista et al., 1973 ; Uhl and Lewis, 1974; Dennis et al., 1969).

llV

PTP

FIG. 5. Derivation of pulmonary resistance and compliance from tracings of transpulmonary pressure (PrJ.), volume(V) and flow(I;') usingthe method of yon Neergaardand Wirz (1927b).Changein lung volumedividedby the changein elasticrecoil pressure(AV/APet)equals dynamic pulmonary compliance.Instantaneous pulmonary resistance is obtained by dividing the resistiveportion of PT~'(shaded) by flow (shaded).

Methods of study of airway smooth muscleand its physiology

/

f

259

I sovol u me

V PTP

\

FIG. 6. Isovolumemethod of determining total pulmonary resistance (RL).Division of the change in transpulmonary pressure (APrr) by the change in flow (AI;')yieldsaverageRL over the respiratory cycle.

(c) Passive exhalation method of Comroe et al. (1954). This was originally developed to measure R,. CL is measured as in (a). At a certain expiratory flow (e.g. 0.51./sec) the part of the tidal volume remaining in the lungs is calculated. Division of this volume by CL gives the resistive P r e causing the selected flow. (d) Time constant method during passive exhalation (Mcllroy et al., 1963). Expired volume is plotted against expired flow and the slope of this line (AV/Af') is the time constant, which can be separated into its component parts if another relaxed expiration is made with a linear external resistance added in series. A simplified version has been used to measure R , in anaesthetized, paralyzed patients (Bergman, 1966). One measures the time interval from the beginning of expiration to the point where 0.368 of the tidal volume remains in the lungs, i.e. where about two thirds of the tidal volume has been expired. This is the time constant of the lung and it is numerically equal to RLCL. Division by CL [determined as in (a)] yields RL. (e) Electrical subtraction (Mead and Whittenberger, 1953). To obtain RL a voltage proportional to volume is subtracted electrically from the Pre signal until pressure and flow are in phase, i.e. until no looping is seen on the P r e - I/plot. Conversely, to obtain CL one closes the loop on the Prr-volume (V) plot by subtracting a voltage proportional to flow. Of the methods measuring RL and CL this is probably the most widely used. A computer version has been validated by Drazen et al. (1976a). (f) Calculation of RL from the area of the pressure-volume loop (i.e. work of breathing). If P r e contained only the elastic component (Pez), the plot of Pry - 17would be a straight line; what makes it loop is the resistive component also contained in Pre. The area of the loop is therefore related to RL. The mathematical background for conversion of area to RL was given by Otis et al. (1950), the method was developed for practical use by Nisell and Ehrner (1956, 1958), Jaeger and Otis (1964) and others, and computer versions have been developed by Smidt et al. (1975) and Jordanoglou et al. (1976). (g) The preparation of Konzett and R6ssler (1940). This method is still popular because ofits simplicity, especially for screening drugs by pharmacologists. In the original version the animal was ventilated with a constant pressure and the volume which did not enter the animal up to that pressure ('overflow volume') was measured. 'Resistance to inflation' caused an increase in overflow volume. Clearly, overflow will increase not only with an increase in RL ('narrow tubes') but also with a decrease in CL ('stiff lungs'), so the method does not discriminate between RL and C~., although at low frequencies it will measure predominantly CL and at high frequencies predominantly RL. Presently the variable measured is more often the swing in Pn, at a constant volume of ventilation.

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1.1.3. Method of forced oscillations 1.1.3.1. Principle. The method was originally used without a measurement of Pv,, i.e. R,~ (not Rt,) was obtained. Sinusoidal oscillations of pressure and flow, generated by a piston pump or by a loudspeaker coupled to a sine wave generator and amplifier, were applied to the subject during breath holding (DuBois et al., 1956b). In later versions oscillations were superimposed on the normal breathing pattern (Mead, 1960a) or were applied to the outside of the thorax which was enclosed in a plethysmograph (e.g. Wohl et al., 1969). Changes in Pao and 1? were measured and the resistive component of Pao was related to 12. Advantages of this method are: (a) no cooperation is required, so the method can be used in children (Wohl et al., 1969 ; Mansell et al., 1972; Cogsweil, 1973 ; Aronsson et al., 1977), anaesthetized patients (Crago et al., 1972; Bergman and Waltemath, 1974) and anaesthetized animals (Gold et al., 1972); (b) oscillations can be applied at all lung volumes (Macklem and Mead, 1967; Woolcock et al., 1969 ; Vincent et al., 1970); and (c) no oesophageal catheter has to be swallowed. Disadvantages are: (a) not only tissue but also chest wall resistance is included, both irrelevant to measurements of airway smooth muscle constriction, Cogswell (1973) found R,~ to be 30 per cent higher than Raw in children ; in 41 healthy adults, R,~ was 2.3, Raw only 1.3 cm H20/I. sec (Fisher et al., 1968); (b) a measurement of lung volume is not included; (c) high oscillation frequencies increase demands on the frequency response of the equipment, especially in children who have a higher natural breathing frequency, i.e. oscillation at even higher frequencies is required to get a clear separation of signals; and (d) the overall accuracy of the method is less than can be obtained with plethysmography or by subtraction methods (Landau and Phelan, 1973). 1.1.3.2. Techniques to select the resistive component of Pao. Due to the higher frequencies involved, we have to consider not only the elastic and resistive but also the inertial component of the total pressure measured, i.e. the component needed to accelerate the air column and tissues. In principle one can 'extract' the resistive component either by oscillating at the resonant frequency of the respiratory system or by artificially creating the condition of resonance. (a) Oscillating at the resonant frequency (DuBois et al., 1956b). During sinusoidal forcing, elastic and inertial pressure components are exactly 180° out of phase, i.e. always opposite in sense. Elastic impedance dominates at low cycling frequencies; inertial impedance dominates at high cycling frequencies. At one intermediate frequency, the resonant frequency, they are exactly equal in magnitude and therefore cancel each other. At this frequency only flow resistive pressure is measured and can be related to flow. (b) Method ofGrimby et ai. (1968). Artificial resonance is created by subtracting electrically from the pressure signal a signal proportional either to volume (at low frequencies) or to volume acceleration (at high frequencies) until pressure and flow are in phase. Grimby et al. (1968) demonstrated a decrease in R,s with increasing cycling frequency in patients with airway obstruction. (c) Method of Goldman et al. (1970). Instants of zero acceleration at the extremes of flow, when flow has stopped rising and before it has begun to fall, occur midway in each phase (inspiration and expiration) and therefore occur at points of equal volume where elastic pressures must be the same. Since neither inertial nor elastic properties can account for any pressure difference measured between these two instants, this difference must relate solely to the flow resistance of the system. Landau and Phelan (1973) compared the two methods in patients with obstructive lung disease. Pressure and flow were so markedly out of phase that unusually low pressure changes (and therefore low R,s values) were measured with the Goldman technique. This was due to time constant inequalities, so points of zero flow at the mouth did not represent points of zero flow in the various compartments of the lungs with varying time constants. Grimby's technique gave somewhat higher values of R,~ but the authors estimated that they still did not represent the true resistance. Several computer versions of the Goldman technique are available (Hyatt et al., 1970;

Methods of study of airway smooth muscleand its physiology

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Stanescu et al., 1975; Aronsson et al., 1977). Watson et al. (1975) developed an automated system using a piston pump instead of a loudspeaker. A piston pump will keep the oscillation volume constant, but a loudspeaker will not. Many authors combine the application of forced oscillations with a measurement of Pr~,, to obtain Rt. (e.g. Vincent et al., 1970; Drazen et al., 1976b). 1.1.4. Interrupter technique (yon Neergaard and Wirz, 1927b) 1,1.4.1. Principle. Originally only Pao was measured: a brief (0.1 sec) interruption of air flow led to momentary equilibration of pressures throughout the bronchial tree, so Pao = Pa~v"The difference between Poo during flow and interruption, related to flow just before interruption was believed to give Ra~ (von Neergaard and Wirz, 1927b; Vuilleumier, 1944). It has since been shown that Patv before interruption is different from Pa~vmeasured during interruption and that the method determines RL plus a small and variable contribution from chest wall resistance (Mead and Whittenberger, 1954). Resistance measured by interrupter was found to be 11 per cent (Clements et al., 1959) to 18 per cent (Mead and Whittenberger, 1954) higher than R~. Using certain extrapolation procedures Jackson et al. (1974) were able to obtain values close to Ra~. When Pre is measured instead of Pao the method determirles R L. The prediction of Clements et al. (1959) that due to slower pressure equilibration the method would lose accuracy in disease was confirmed by Lloyd and Wright (1963). 1.1.4.2. Modifications of the technique. (a) Interrupter technique with measurement of Poo. Vuilleumier simplified the original method by using only one transducer to measure flow (before interruption) and Poo (during interruption), In the device developed by Clements et al. (1959) two motor driven concentric rotating tubes with special valving produced 10 interruptions/sec. Resistance could be measured even at turbulent flow rates. (b) Interrupter technique with measurement of Pre. In recent years the interrupter principle has been used in conjunction with pleural or oesophageal pressure measurements to determine RL (e.g. Coon et al., 1975; Ingram, 1975). Some authors control flow during measurements, either with special regulator valves (Allander et al., 1964; Jonson, 1969) or with an external resistance consisting of a rubber stopper with a hole (Colebatch et al., 1973). Colebatch et al. (1973) related pulmonary conductance (GL) to Pet- The GL-Pe~ relationship was undisturbed in some patients with emphysema, i.e. in this condition bronchial narrowing was due to reduced elastic recoil, not to a change in intrinsic bronchial properties. The relationship was disturbed in asthmatic patients, indicating a change in bronchial, not parenchymal, properties. Bronchodilator treatment produced a more normal slope. GL-Pet plots can thus help to distinguish between bronchial narrowing due to smooth muscle contraction and due to lack of recoil. A computer-aided method to process all the measurements and construct the plots is available (Colebatch et al., 1978). This indirect influence of Pet on resistance (through a change in airway dimensions) must not be confused with the direct influence of Pe~ (as driving pressure) on maximal flow. 1.1.4.3. The 'additive' method (Dirna#l, 1953). A description of this method in English is given by Kures (1974). Instead of alternating between free airflow and complete interruption one alternates between two different resistances, R 1 and R 2, each attached to one limb of a Ypiece with a shutter alternating between the two limbs. Both situations can be described by two simultaneous equations: Potv = (Raw + R l)" V1 P~tv = (Raw + R2)" f'2. These equations eventually lead to the simple solution: Ra~ = (P2 - PI)/(I;'I - I;'2), where P1 and P2 are the pressure differences across the added resistances, easily measured as mouth pressure. No significant difference was found between resistance values measured by this method and by plethysmography, both in adults (Sobol, 1970) and in children (Kures, 1974). The basic assumption that Pa~vremains the same in the two alternative situations has not yet been proven but it could well be that the error is smaller than with the conventional

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H.L. HAHN and J. A. NADEL

interrupter technique because volume (tissue) movement is never stopped completely, so the contribution from tissue resistance should be less. 1.1.5. Comparison of different methods for measuring resistance Lloyd and Wright (1963) found R~wmeasured by plethysmography to be the most sensitive and interrupter resistance using the Clements valve to be the least sensitive means of detecting bronchoconstriction induced by dust particles; several maximal flow indices ranged in between. In a similar comparison by Frank et al. (1971) the absolute magnitude of changes measured by four methods varied, but when corrections were made for differences in volume history, cycling frequency and the variability of each method, three of the methods (plethysmography, subtraction method, forced oscillations) had comparable sensitivity; interrupter resistance was less sensitive and also most variable. Bergman and Waltemath (1974) measured R,s in anaesthetized, paralyzed subjects using four different subtraction techniques and a method of forced oscillations. They found systematic, but small, differences between the five methods, the highest R,s values being derived from analysis of passive exhalation. The isovolume technique was least sensitive in detecting an external resistor in series with the respiratory system. 2. MEASUREMENT OF INDICES OF MAXIMAL FLOW (l?m~) These are the variables most frequently used in clinical practice ;they are simple to perform, but their theory is complex. 2.1. PRINCIPLE OF THE MEASUREMENT

In normal subjects flow during maximal expiration is limited by dynamic compression of the central, intrathoracic airways (Fig. 7). The driving pressure for expiratory flow is alveolar pressure (P,~v), the sum of pleural pressure (Ppt) and the elastic recoil pressure of the lung (Pc,), both of which are positive during forced expiration: P,Iv = Pe~ + Ppl. From Polv mouthwards airway pressure falls until it reaches atmospheric pressure at the mouth (Pa, = 0). In the airways near the alveoli, intraluminal pressure is greater than Pp~; in those near the thoracic outlet, Pp~ exceeds intraluminal pressure, so these airways are compressed. Increasing effort, although increasing driving pressure, also increases Ppt, i.e. causes dynamic compression. Two factors add to this increase: firstly, turbulent flow develops, which increases the pressure drop down the airways. Secondly, the decrease in total cross-sectional area of the airways between alveoli and large airways (Fig. 3) increases the linear velocity of

Segment S -------~J, I-D-~

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FIG. 7. Dynamic compression of airways. During forced expiration airway pressure falls from alveolar pressure (Pa~v) which is the sum of the elastic recoil pressure (Pel) and pleural pressure (Ppl) to zero pressure at the airway opening (P,,o). At the equal pressure point (EPP) intraluminal equals extraluminal, i.e. pleural pressure (Ppl) Airways downstream from EPP will be compressed because intraluminal pressure is less than P~. The S-segment is the airway segment extending from alveoli to the point of dynamic compression~ the D-segment that extending from the point of compression to the mouth.

Methods of study of airway smooth muscle and its physiology

263

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the airstream, reducing the pressure within the airway lumen (Bernoulli effect). As a result, increments in Pa~v produce progressively smaller increments in expiratory flow until eventually flow reaches a plateau and thereafter is independent of the pressure generated. This is best seen from a plot ofP, zv against expiratory flow at one particular lung volume, an isovolume-pressure-flow (IVPF) curve (Fig. 9). An IVPF curve removes one variable (lung volume), which itself has considerable influence on airway resistance. A series oflVPF curves obtained at different lung volumes (Fig. 9) shows that plateaux of l?m,xOccur at lung volumes below about 70 per cent of the vital capacity (VC). Tests of forced expiration are therefore independent of effort over most of the VC, provided the minimum alveolar pressure needed to reach a flow plateau is generated. The variables contributing to flow limitation have been analysed (e.g. Mead et al., 1967 ; Pride et al., 1967; Jones et al., 1975a, 1975b). Since intraluminal (lateral) airway pressure is greater than Pvz close to the aiveoli but smaller than P~ in the compressed segment, there must be a point in between where intraluminal equals pleural pressure; at this equal pressure point (EPP) (Mead et al., 1967) transmural pressure, (Ptm, intra- minus extra-luminal pressure), is zero. With increasing expiratory effort, the EPP moves upstream towards the alveoli. When expiratory flow no longer increases with increasing effort the EPP has become fixed and the segment from alveoli to EPP (upstream segment) remains constant at a given IVPF-CURVES

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FIG. 9. Is,volume-pressure-flow (IVPF) curves at different lung volumes expressed as a percentage of the vital capacity (per cent VC) and maximum expiratory flow-volume (MEFV) curve. The MEFV summarizes some of the information contained in a family of IVPF curves, where each single IVPF curve describes the relationship between driving pressure = alveolar pressure (P, tv) and flow at one particular lung volume. Dashed horizontal lines show h o w the flow rate on the MEFV curve corresponds to the maximum flow rate on the corresponding IVPF curve.

264

H . L . H A n s and J. A. NADEL

lung volume (Fig. 7). The driving (inflow minus outflow) pressure across the upstream segment equals Pe~. The resistance of the upstream segment (R,~) during l?,,ax is therefore given by R,s = Pe~/fz,~x and l?max = PeflR,,. Thus Mead et al. (1967) analyzed l?m~xin terms of elastic recoil pressure and upstream resistance, which does not necessarily identify the actual cause of flow limitation. If, for instance, the downstream segment becomes collapsible, causing a mouthward shift of the EPP by blocking its further upstream migration with increasing effort, in Mead's analysis this will increase apparent upstream resistance by lengthening the upstream segment. The possible influence of compliance changes in the airways that limit flow was taken into account by Pride et al. (1967) who focused on the flow limiting segment of the airway. When Pp~ exceeds the pressure within the collapsible airway by a sufficient amount for it to narrow critically and limit flow, the point of narrowing divides the airway into two segments: the S-segment from alveoli to the point of critical narrowing and the D-segment from this point downstream to the mouth. Expiratory flow will reach a maximum when the resistive pressure drop down the S-segment equals (Pet - Ptm'), where Ptm' is the value of Ptm at which the airway narrows to control flow. Negative Ptm, indicates an airway resisting collapse, positive Ptm' would be found in an airway collapsing even at positive (distending) pressures. The resistance of the S-segment (Rs) is given by Rs = (Pel Pt,~,)/(/~ and V~ax = (Pet - e,~')/~. This equation shows that l?ma~ depends not only on the dimensions of the airways (as Raw does) but also on elastic recoil pressure (Pe~), the driving pressure through the upstream segment (Fig. 7) and on the collapsibility of the airways undergoing compression (P,~,). Jones et al. (1975a, 1975b) analysed the effect of airway compliance on 17~ and demonstrated that under certain experimental conditions 17,~ was determined by (and could be predicted from) the compliance of a compressed segment of trachea alone. Dependence of l;'mo~ on local airway properties at the 'choke point' is also predicted by the 'sound wave hypothesis' (Dawson and Elliott, 1977; Elliott and Dawson, 1977) which proposes that flow limitation occurs when flow velocity equals the speed of propagation of pulse waves, l?,o x is determined by the cross-sectional area and stiffness ofthe airway at the choke point, Ptm, gas density and velocity profile. It should be noted that, in contrast to the measurement of R ~ (Fig. 3), l?mo~ is not affected by the resistance of airways mouthward from the compressed segment (e.g. larynx). In normal subjects during l?m,x, compressed segments are in trachea and lobar bronchi over most of the VC, but move peripherally below FRC and in disease. An increase in smooth muscle tone can affect all of the variables contributing to flow limitation : increased tone in peripheral airways can increase elastic recoil pressure (Nadel et al., 1964; Colebatch et al., 1966; Woolcock et al., 1969b), tending to increase I?max. It will certainly increase airway resistance, tending to decrease 17~. Increased smooth muscle tone may increase (Murtagh et al., 1971) or decrease (Olsen et al., 1967a, 1967b; Palombini and Coburn, 1972) airway collapsibility, which could affect 17 . Although in most circumstances an increase or a decrease in upstream resistance will be the main effect of a change in smooth muscle tone and will determine how I?=~ changes, only additional measurements (e.g. Pe~, Ro~) will enable the investigator to quantify the contribution of each variable. 1.2.2. Tests of maximal expiration Maximal expiration can be recorded as volume expired over a given time (1.), as a time taken to exhale a given volume in sec (only rarely used and will not be considered), as an instantaneous flow rate (l./sec) and as a plot of instantaneous flow rate against lung volume (maximal expiratory flow volume (MEFV) curve or partial expiratory flow volume (PEFV) curve). 1.2.2.1. Timed expired volumes. If change in volume is plotted against time during a maximally rapid exhalation from total lung capacity (TLC) to residual volume (RV), the forced vital capacity (FVC) is obtained (Fig. 8). Various segments of the FVC can be measured. The volume expired in the first second is the FEV 1 ; comprising about 75 per cent of the FVC it is relatively independent of effort. Shorter (FEVo.75, FEVo.~) and longer (FEV2,

Methods of study of airway smooth muscle and its physiology

265

FEV3) timed capacities have been used, sometimes after discarding initial volumes, but these measurements have not gained similar popularity. 1.2.2.2. Recordings of instantaneous flow rate. Peak expiratory flow rate (PEFR, PEF) or peak flow (PF), the highest flow rate during a forced expiration, is measured with a pneumotachograph or other flow measuring device such as the portable Peak Flow Meter (Wright and McKerrow, 1959). Although effort dependent and not as sensitive as some other variables, the measurement is easily learned and well reproduced by patients. The mean midexpiratory flow rate (MMF, MMFR or MMEF, Leuallen and Fowler, 1955) is usually obtained from the spirogram (Fig. 8) by computing the average flow rate over the middle half (25-75 per cent) of the expired volume. It practically excludes effort-dependent flow and is recorded accurately even by equipment too slow to give a true record of the initial fast portion of the spirogram. 1.2.2.3. Maximum expiratory flow-volume (MEFV) curve (Fig. 9). Instantaneous flow is plotted against change in volume, measured as expired volume at the mouth or as TGV in a volume plethysmograph. Addition of a time-pulse generator (Hankinson and Lapp, 1970) will provide FEV t from the same manoeuvre. PEFR and M M F can also be obtained from this curve (Fig. 9); M M F corresponds closely to lYmo~at 50 per cent VC (MEFs0 or l?moxso).It will decrease earlier than the FEVt or PEFR during induced bronchoconstriction. Due to the increase in RV that usually accompanies induced bronchoconstriction, MEFso will actually be measured at higher absolute lung volumes. The flow rate at the same absolute lung volume, e.g. at TLC-60 per cent, will therefore be even more sensitive in detecting induced bronchoconstriction (Fig. 10, Bouhuys et al., 1969 ; Olive and Hyatt, 1972), but, of course, a measurement of absolute lung volume by plethysmography is required, making the test more complicated. A further increase in sensitivity was reported by Nadel (1970) who showed that airway smooth muscle contraction was detected more sensitively the smaller the inspired volume. This observation was confirmed by Bouhuys et al. (1969) who recorded I/m=xat TLC-60 per cent not during expiration from TLC but during expirations from midVC, i.e. during partial expiratory flow-volume (PEFV)-curves (Fig. 10).

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266

H.L. HAHNand J. A. NADEL

1.2.3. Tests designed to distinguish between the variables contributing to flow limitation 1.2.3.1. Maximalflow static recoil ( M F S R ) curve, f/r,,~ is plotted against Pel. The slope of this plot has the dimensions of conductance (l./sec cm H 2 0 ) and it represents the conductance of the segment of the airways along which the pressure drop equals Pet (upstream conductance in Mead's analysis). If a reduction in l?ma~ is entirely due to loss of recoil, the MFSR curve will have a normal slope but will be foreshortened : the flow produced per unit recoil is normal, but there is less recoil. Decrease in upstream conductance will be indicated by a smaller slope : elastic recoil pressure is normal, but the flow produced per unit recoil is decreased. Finally, a parallel shift to higher Pet (positive intercept on Peraxis, indicating zero conductance at positive distending pressure) is due, at least in part, to an increase in P,.,, i.e. increased collapsibility of flow limiting airways (Pride et al., 1967; Freedman et al., 1975). 1.2.3.2. Influence of viscosity and density. Schilder et al. (1963), examining the effect of breathing gases of differing viscosity and density, noted that the lower third of the MEFV curve was most strongly affected by gas viscosity whereas the upper third was most strongly affected by gas density. Wood and Bryan (1969) likewise noted the effect of gas density on the upper three quarters of the MEFV curve. This is because in the smaller airways which presumably determine flow at low lung volumes, flow is laminar and therefore dependent on gas viscosity but not on gas density. The reverse is true for the larger intrathoracic airways whose diameter determines f'r,a~ at high lung volumes. When peripheral airways are constricted, the usual increase in l?m°x on changing from breathing air to breathing a mixture of low density (80 per cent helium-20 per cent oxygen) is reduced (Despas et al., 1972; Dosman et al., 1975). Since breathing H e - O 2 has negligible effects on elastic recoil (Hutcheon et al., 1977), the increase in flow on He-O 2 may specifically indicate an increase in upstream conductance (Wood and Zeismann, 1976) but a shift of the flow limiting segment to the periphery is also possible (Mink and Wood, 1978). Ways to quantify the 'helium response' are shown in Fig. 11. 1.3. OTHER FUNCTIONAL MEASUREMENTSREFLECTING AIRWAY CALIBRE 1.3.1. Dead space method Fowler (1948) introduced the gas analysis method for N2 and Young (1955) modified it for CO2. In principle one measures the volume of air which has left the lungs up to the time when the front of CO2-rich gas from the alveoli reaches the sampling point. Corrections have to be made for the fact that the CO 2 front is not rectangular and for the lag and response time of the CO2 analyzer. The method has been used to assess constriction in large airways (Severinghaus and Stupfel, 1955; Nadel et al., 1964; Green and Widdicombe, 1966), airway hysteresis (Froeb and Mead, 1968) and airway compliance (Olsen et al., 1967a). 1.3.2. Balloons in airways; recordings from isolated segments The principle of inserting a balloon into the trachea or into a bronchus and recording changes in airway circumference as changes in balloon pressure and volume is old. Roy and Brown (1885) found that the pressure in an endobronchiai balloon increased with asphyxia and that this was preventable by vagotomy. Ellis and Livingstone (1935) inserted a brass tube with an outer cuffclose to its tip (similar to an endotracheal tube) into a bronchus so the lung distal to the balloon was not obstructed. The variable recorded was the volume change in the cuff. A more recent modification was the method of Coigan (1964). His was a cylindrical balloon of fixed length (rigid end plates), attached to the outside of a tube. It was placed in a bronchus with an internal diameter just sufficient to contain the balloon and the balloon volume was known. Bronchoconstriction induced by i.v. histamine caused volume displacement from the balloon (measured as change in pressure) and this allowed calculation of bronchial diameter using the cylinder formula: V = (D/2) 2 • L where V = cylinder volume, D = cylinder diameter and L = cylinder length. Since cylinder shape probably changed somewhat this was only anapproximation. Even so, knowing the pressure inside the

Methods of study of airway smooth muscle and its physiology

267

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bronchus (from balloon pressure) and the pressure surrounding it (Ppt), they could calculate transmural (bronchial minus pleural) pressure. Related to the change in diameter, this value gave bronchial compliance, which they found to decrease after histamine. Palombini and Coburn (1972) similarly noted a decrease in compliance of a liquid-filled segment of trachea after vagal stimulation. Isolated segments of trachea (with or without a balloon) have been used extensively in studies of reflex bronchoconstriction (Kahn, 1907; Loofbourrow et al., 1957; Nadel and Widdicombe, 1962a; Green and Widdicombe, 1966; Graf et al., 1975) and as in vivo pharmacological preparations to assess drug effects (Lynn-James, 1969). This method has also been combined with measurements of the resistance of the perfusing blood vessels where it was found that fl-adrenergic receptors dominated in tracheal, alphaadrenergic receptors in vascular smooth muscle (Himori and Tairi, 1976). 1.3.3.Airway catheters and needles

These are used to measure the longitudinal pressure drop along the bronchial tree, to determine the resistance of segments of the bronchial tree (at low flows) or to locate equal pressure points (during maximal flow). The latter was first done by Koblet and Wyss (1956) with liquid-filled end-hole catheters. Macklem and Wilson (1965) used side- and end-hole catheters to measure lateral and total bronchial pressures, respectively. By pulling catheters

• ~"~'~'~

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FIG. 12. The retrograde catheter technique, The retrograde catheter is a piece of polyethylene tubing, bell shaped at one end and is shown in its final position (from Macklem and Mead, 1967). JPT Vol. 7, No. 2 - - E

268

H. L. HAHN and J. A. NADEL

back known distances they located EPP’s in segmental and larger bronchi. Catheters have been used to follow the mouthward shift of EPP after surgical weakening of the trachea (Herzog et al., 1968) and to follow the opposite shift toward alveoli during vagal stimulation (Pride et ai., 1964). Hyatt and Wilcox (1961) partitioned resistance by measuring lateral tracheal pressure with a needle inserted 2 cm below the larynx, finding that almost half the total airway resistance was located in extrathoracic airways. While these (and many other) authors partitioned resistance fairly high in the bronchial tree, the use of retrograde catheters, introduced by Macklem and Mead (1967), made it possible to assess the contribution to R,, of quite small airways which would become obstructed by anterograde catheters (Fig. 12). A piece of piano wire is used to pull a polyethylene catheter with a bell at the end through airways, parenchyma and pleural surface until the bell of the retrograde catheter becomes wedged in a bronchus (Fig. 12). Pulmonary resistance can then be partitioned into two components : one between the retrograde catheter and the alveoli (peripheral resistance, RP) and one between the trachea and the retrograde catheter (central resistance, R,). If a silk suture is tied around the catheter bell, this can be pulled towards the trachea measured distances and resistance can be partitioned at more than one point (Macklem et al., 1969). Retrograde catheters have also been used in maximal flow studies to follow the upstream shift of EPP during vagal stimulation (Gardiner et al., 1974) and they have been implanted chronically in dogs (Battista et al., 1973); this will evoke a foreign body reaction around the tip. Several problems with the retrograde catheter (airway obstruction, distortion of surrounding parenchyma, the fact that the catheter will not ‘recognize’ pressure losses due to convective deceleration and acceleration) have been discussed (Macklem and Mead, 1967). Other problems exist : at high lung volumes R, is extremely small, while the tissue resistive component increases sharply with Pet, leading to an overestimate of R, especially when oscillation methods are used (Hildebrandt, 1971; Hoppin et al., 1978). Also, since at high lung volumes a large pressure component due to elastance has to be subtracted from a small component due to resistance to ‘close the loop’ any phase lags in the catheter-transducer system will also introduce errors (Hoppin et al., 1978). Some reports of increasing R, at high lung volumes are probably invalid. The use of multiple retrograde catheters through which the lung could be kept inflated at a constant volume during expiratory flow measurements was first reported by Culver et al. (1973) and modified slightly by Hughes et al. (1974). 1.3.4. Other intrabronchial or peribronchial devices Schwieler (1966) measured variations in bronchial diameter in a qualitative way with a capacitive transducer, making use of the fact that the capacitance between a small metal spiral situated in a bronchus and the bronchial wall changes with bronchial diameter. Souhrada and Dickey (1976) sutured a special extraluminal strain gauge (described first by Bass and Wiley, 1972) on the outside of the trachea of guinea pigs and placed the cable so it emerged dorsally in the neck following closure of the incision. They were able to record spontaneous changes in tracheal diameter in awake animals. Akasaka et al. (1975a, 1975b) obtained bronchial electromyograms in viuo by inserting a flexible polyethylene catheter with a bipolar platinum ring electrode close to its tip into a bronchus under fluoroscopic control. By pulling a string they could bend the tip, bringing the ring electrodes in close contact to the wall. Comparison with recordings obtained with a direct peribronchial needle electrode (which had been used earlier by a number of authors) showed similar discharge patterns, proving that the mucus membrane was not a serious barrier to the recording technique, and EMG potentials were easily distinguishable from EEG patterns. They recorded the EMG of airway smooth muscle during induced bronchoconstriction in asthmatic patients (1975b).

1.4. MORPHOLOGICAL METHODS Again, changes in tone are inferred from changes in airway calibre. Although in general morphological methods are a more direct means of doing this than the functional methods

Methods of studyof airwaysmoothmuscleand its physiology 45

cm

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FiG. 13. Photographof alveolarsacs and ductsfromfoetalrabbit lungs(fromEnhorning, 1977). discussed so far, some of these methods measure inner lumen width and will therefore be influenced by changes in mucosal thickness. 1.4.1. Photography, kinematography, bronchoscopy, microscopy Knudson and Knudson (1973, 1975) and Jones et al. (1975a, 1975b) illuminated a 2 mm segment of trachea from the outside with a slit lamp and photographed the illuminated ring of trachea through a perspex window at the mouthward end of the trachea in excised lungs. Flow was maintained by means of a side tube between the perspex window and the end of the trachea during maximal flow studies. Jones et al. (1975) extended this to the main bronchi by attaching a fiberoptic bronchoscope to the camera or to a cinecamera running at 60 frames/sec to follow the rapid change in tracheal and bronchial area during maximal flow. Acetylcholine reduced tracheal area but made it less compressible at negative Ptm. Tracheal collapse has also been observed and filmed through rigid bronchoscopes by a number of investigators (e.g. Gandevia, 1963; Herzog et al., 1968). Macklem and Mead (1968) confirmed the location of EEP (obtained from catheter studies) by taking slow motion movie films of excised lungs during forced deflations: downstream from EPP the posterior membraneous sheath was invaginated and upstream it was distended. Vidruk et al. (1977) used a flexible fibrebronchoscope to apply acetylcholine and histamine aerosols locally to airways through a 1.6 mm catheter advanced through the inner channel of the bronchoscope. They were able to observe bronchoconstriction after both drugs, but only histamine regularly stimulated sensory receptors in the airways. Enhorning (1977) obtained beautiful photographs (Fig. 13) of alveolar sacs and ducts from rabbit fetal lungs by placing

270

H.L. HAHNand J. A. NADEL

excised lungs in a saline bath and mounting them with their diaphragmatic surface turned upwards against a glass plate just below the surface. The glass plate was in the focal plane of a camera. Two lungs (control and treated) were mounted edge to edge with a scale in between indicating inflation pressure. Lighting came from a flashlight mounted below the saline bath. For the same purpose, i.e. to study the most peripheral airways of the lungs, methods have been developed to freeze lungs rapidly with liquid propane, cut them in blocks in the cryostat, freeze-dry them and obtain thin or thick slices. The size of respiratory bronchioles, alveolar ducts and alveolar sacs can then be measured under a stereomicroscope with an image splitting eye piece (Staub and Storey, 1962). Right heart injections of histamine and BaSO4 microemboli constricted respiratory bronchioles and alveolar ducts by smooth muscle contraction (Nadel et al., 1964; Colebatch et al., 1966). !.4.2. Fluoroscopy, roentgenography and bronchography Air bronchograms have long been used. Macklin (1925) observed the lengthening and widening of bronchi with respiration by comparing inspiratory with expiratory roentgenograms of the chest. Dekker and Ledeboer (1961) observed tracheobronchial compression fluoroscopically during voluntary wheezing expiration and they quantified their observations by obtaining air tracheograms and bronchograms, tracing outlines on transparent paper and reproducing them photographically on a smaller scale so they could be mounted side by side. Murtagh et al. (1971) demonstrated bronchial closure in excised dog lungs after application of methacholine. Although air bronchograms have the advantage of being obtained noninvasively and of not altering surface properties of the airways, the range of bronchi observable with this technique is limited in vivo and contours are often not outlined with sufficient sharpness. This led to the use of aqueous or oily iodine contrast media which could be instilled in the bronchial tree through catheters or tracheal puncture. From such bronchograms, Stutz (1950) calculated the total cross-sectional area of the airways for several bronchial generations. He suggested the high values for peripheral resistance predicted from earlier studies (based on collapsed lungs) were incorrect. Kilburn (1960) used aqueous propyliodone in dogs and observed bronchoconstriction when the animals breathed 5 per cent CO2. Following right heart injections of smooth muscle constricting agents, lobar and segmental bronchi constricted more than trachea and main bronchi. Marshall and Holden (1963) noted the greater compliance of small (less than 1.7 mm) compared to larger bronchi. When used in vivo, these contrast media had the disadvantage of being toxic in sensitive subjects (iodine), interfering with gas exchange by partial or total occlusion of the lumen and sometimes irritating airways. Moreover, they could only be applied through special catheters and gave no details of the mucosa. Improvement came with the introduction of powdered tantalum (Nadel et al., 1968), which is not toxic, outlines bronchi without obstructing them, gives much better mucosal detail down to I mm airways, is more radiopaque (i.e. less substance is required) and can potentially be inhaled. In practice, inhalation techniques through the mouth have met wi~h limited success because large amounts of the medium are deposited in the oropharynx and are then swallowed (Strecker et al., 1974). Also most of the material which enters the bronchial tree goes to the dependent lung regions, making inhalation in multiple body positions necessary (Smith et al., 1976). Disadvantages are that tantalum dust (like any dust) can burn when aerosolized in 0 2 in the presence of a spark (Goerg et al., 1973), so compressed air, not pure oxygen must be used and the equipment must be grounded electrically. All contrast materials in contact with the airway mucosa may cause bronchoconstriction unless atropine is given (Strecker et al., 197~): The size range of airways that can be studied by tantalum bronchography has been further widened by the use of a microfocal spot X-ray tube with primary 5-10 fold magnification, allowing examination of airways well below 1 mm (Nadel et al., 1971) (Fig. 14). Bronchography has also been performed with powdered iodine contrast media (Fischer and Blaug, 1969; Parrisius et al., 1972) and powdered lead, a toxic material (Leopold and Gough, 1963). Cinebronchography with liquid contrast media has been used by several authors, e.g. by Holden and Ardran

Methods of study of airway smooth muscle and its physiology

271

FIG. 14. Tantalum bronchogram from a dog, obtained with a microfocal spot X-ray tube (0.06 mm OD) with primary 5-10 fold magnification.

(1957) who disproved bronchial peristalsis. Jones et al. (1975) examined the flow limiting segment in the canine trachea using tantalum cinebronchograms. Bronchographic techniques suffer from problems relating to magnification. Magnification is negligible in bronchi close to the film but increases with increasing distance from the film. Another problem is the measurement of bronchial length which is unreliable because of foreshortening. To overcome these geometric errors, Hughes et al. (1972) used double-image (stereoscopic) X-ray exposures to measure length and diameter. In a few instances, Hughes et al. (1974) analysed stereoscopic pairs of X-rays on a stereocomparator for bronchial crosssection (shape). However, stereo techniques are time consuming and have only been used in excised lungs. 1.5. SPECIALPROBLEMSENCOUNTEREDIN STUDIESIN VIVO 1.5.1. Volume history A given intervention has different effects on airway smooth muscle depending on the breathing manoeuvre preceding it. Nadel and Tierney (1961) noted that the bronchoconstriction induced in normal subjects (e.g. by cigarette smoke) was reduced for 1-2 min following a deep breath. In anaesthetized dogs, vagal stimulation causes greater bronchoconstriction at a given Pre when this Pre is reached during inflation from RV than during deflation from TLC and only during inflation from-RV will an increase in smooth muscle tone increase lung elastic recoil (Yoshida, 1964; Woolcock et al., 1969; Hahn et al., 1976). This can also be shown in excised lungs (Hughes et al., 1975 ; Clay et al., 1977). According to some authors, even normal, resting tone can be reduced temporarily after a full inflation (Vincent et al., 1970; Green and Mead, 1974). Volume history has to be considered when different measurements are used to assess the effect of the same intervention. The discrepancy between an 89 per cent increase in RL and an only 7 per cent decrease in peak flow was suspected to be due to the difference in volume history, peak flow being measured following a maximal inspiration and RL during quiet

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H.L. HArtNand J. A. NADEL

breathing (Frank et al., 1962). Differences in volume history are likely to be responsible for the greater sensitivity of PEFV than MEFV manoeuvres (Bouhuys et al., 1969) and even for differences between two successive PEFV manoeuvres with no full inspiration in between (Wellman et al., 1976). Opposite changes, i.e. an increase in Row after rapid inspirations, was demonstrated in some normal subjects by Lloyd (1963) but has been more commonly encountered in asthmatic patients (Butler et al., 1960; Simonsson et al., 1967; Gayrard et al., 1975). Causes may be stimulation of rapidly adapting sensory receptors in the airways (Lloyd, 1963; Simonsson et al., 1967), release of prostaglandins, or changes in the contractile mechanism of airway smooth muscle (Orehek et al., 1975b). 1.5.2. Route o f administration o f bronchoactive substances Intravenous acetylcholine was 2.5 times less effective than i.v. serotonin in causing bronchoconstriction (Pun et al., 1971) but, given as an aerosol, was about ten times more potent than serotonin (Sampson and Vidruk, 1975; Hahn et al., 1978), probably because acetylcholine is destroyed rapidly by acetylcholin-esterases in the blood. Intravenous histamine releases catecholamines from the adrenal medulla (Stascewska-Barczak and Vane, 1965), which may decrease the effect of histamine on peripheral airways in cats (Colebatch and Engel, 1974b) and man (Ploy-Song-Sang et al., 1978). The hypotension that follows injection of histamine may lead to baroreceptor-mediated increases in sympathetic efferent activity (Diamond, 1972). Similar effects have to be considered for all drugs affecting blood pressure. Prostaglandin F2~ can also facilitate catecholamine release from the adrenal medulla (Brody and Kadowitz, 1974). This effect and the fact that up to 92 per cent of circulating prostaglandin El, E2 and F2~ is inactivated during passage through the lungs (Ferreira and Vane, 1967) may explain the failure of Brown et al. (1978) to demonstrate hyperreactivity to i.v. PGF 2~ in asthmatic patients when hyperreactivity to aerosolized PGF2~ has clearly been demonstrated (Math6 amd Hedqvist, 1975). Similar differences may be found for other mediators which are removed rapidly from the circulation, usually by endothelial cells (Junod, 1977). Another explanation for route-dependent differences in sensitivity may be that aerosolized drugs can stimulate sensory nerve endings in the airways (Vidruk et al., 1977), causing reflex bronchoconstriction.

1.5.3. Central vs peripheral airways Often we want to know whether an intervention affects predominantly large or small airways or both. Before reviewing the various methods to do this, we should consider the following points: (1) Since there is no universally accepted definition of 'central' and 'peripheral' airways, we must always define our terms to avoid confusion. (2) The method of inducing bronchoconstriction may influence the sites in the airways that are affected predominantly. We have seen the importance of route of delivery. With aerosols, particle size, flow rate, lung volume and volume history at the start of inhalation must also be considered (Yeates et al., 1975 ; Dolovich et al., 1976; Ruffin et al., 1978). (3) When a 'new' physiologic method claims to differentiate between narrowing of specific airways, structural proof of this localized involvement should be a requirement of validation of the method. We shall review some methods to achieve and to assess selective bronchoconstriction. 1.5.3.1. Bronchography. This is the most direct way to study the longitudinal distribution of bronchoconstriction induced by drugs or nerve stimulation. Using tantalum bronchography Nadel et al. (1971) established that vagal stimulation caused maximum constriction in 1-5 mm bronchi in dogs (0.8-2 mm bronchi in cats) while little constriction occurred in airways less than 0.5 mm wide. Cabezas et al. ( 1971) showed that simultaneous stimulation of the sympathetic nerves was able to reduce but not abolish vagally induced bronchoconstriction in those same airways. After vagotomy, stimulation of the sympathetic nerves (Cabezas

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et al., 1971) or infusion of catecholamines (Hahn et al., 1976) produced no further bronchodilation.

1.5.3.2. Dead space (Vo) and pulmonary resistance (RL) VS pulmonary compliance (CL). Nadel et at. (1964), Olsen et at. (1965) and Colebatch et al. (1966) defined as peripheral airways those perfused by the pulmonary circulation, mainly respiratory bronchioles and alveolar ducts; these airways are not under significant vagal control (Nadel et al., 1971 ; Cabezas et al., 1971 ; Oisen et al., 1965). Larger airways are perfused by bronchial arteries and are controlled by the vagus nerves. The concept was developed that changes in RL and in Vo primarily reflected the calibre of the central airways while changes in CL reflected the calibre of peripheral airways. Thus in cats, irritation of the larynx, which causes vagally mediated reflex bronchoconstriction (Nadel and Widdicombe, 1962a) increased RL 110 per cent and decreased Vo 19 per cent but it did not decrease CL (Nadel et al., 1964). These findings were confirmed in rabbits: vagotomy decreased RL and efferent vagal stimulation increased RL, but neither procedure affected CL (Karczewski and Widdicombe, 1969). On the other hand, right heart injection of BaSO4 microemboli decreased CL 38 per cent, increased Vo 12 per cent and increased RL only 50 per cent, suggesting predominant peripheral bronchoconstriction. The location of airway constriction was confirmed by anatomical studies after rapid freezing and by tantalum bronchography (Clarke et al., 1970). 1.5.3.3. Retrograde catheter studies. 'Peripheral' and 'central' to users of this method means peripheral and central to the retrograde catheter, which is usually wedged in 2-3 mm airways but is sometimes found in larger airways post mortem (Woolcock et al., 1969a, 1969b), and some authors have used catheters with larger (4 ram) bells (Battista et al., 1973; Wood et al., 1976), which will shift the dividing line to airways of larger diameters. Differences in definition have a direct bearing on the interpretation of results. Thus Woolcock et at. (1969a) found that vagal stimulation constricted predominantly central airways in some dogs and predominantly peripheral airways in others. This does not contradict the morphological studies quoted because Nadel et al. (1971) found that vagal stimulation caused striking narrowing of airways 1-5 mm in diameter; thus retrograde catheters were straddling the areas of maximal constriction. 1.5.3.4. Density dependence of maximal flow and resistance. The 'helium response' of maximal flow has been described (see Section 1.2.3.2). It is determined by the relative contribution of turbulent and convective acceleration to flow limitation. If, after a given intervention, density dependence (I?mo,5o, Visol?) increases, this is taken as indicating predominantly central airway constriction; if it decreases, it is taken as predominantly peripheral airway constriction. The measurement is simple and noninvasive, but the definition of'central' and 'peripheral' is not precise; there are no two discrete flow regimes (laminar in some airways and turbulent in others), but flow regimes change continually between alveoli and the point of flow limitation. Using analyses based on density dependence McFadden et al. (1977), studying exercise-induced asthma, observed a predominantly central bronchoconstriction in the group with mild obstruction prior to study and a predominantly peripheral constriction in those with more severe impairment before exercise (lower Pet and MMF). This may suggest that the disease progresses with increasing severity from central to peripheral airways. Lavelle et al. (1978) demonstrated a low helium response in patients with chronic obstructive pulmonary disease while 5 of 6 patients with documented tracheal obstruction had a normal helium response. Density dependence of resistance rests on principles similar to those of maximal flow but has been better validated: thus in dogs Barnett (1967) increased central resistance by placing a clamp on the lower trachea and increased peripheral resistance by i.v. histamine (which decreased CL). Only after tracheal constriction did helium breathing decrease resistance. Similarly in dogs, RL measured by forced oscillations increased with increasing gas density from breathing helium to air to SF6 and virtually all of this was due to an increase in central

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resistance, i.e. the resistance central to a retrograde catheter with a 4 mm bell (Wood et al., 1976). Drazen et al. (1976b) extended this by combining variations in gas density with those in flow: RL central to an anterograde catheter increased from 50 per cent of total RL when the dogs breathed helium-oxygen and RL was determined at 0.251./sec to 63 per cent when the animals breathed air and RL was determined at 0.5 l./sec to 80 per cent while breathing SF6oxygen and when RL was determined at a flow rate of I l./sec. A 'pure' increase in central resistance (banding the trachea) caused a greater increase in RL with SF 6 measured at 11./sec than with He measured at 0.25 l./sec and increased the resistance central to the catheter; i.v. histamine (after atropine) had the opposite effect. 1.5.3.5. Flow rate response vs conductance response. The 'flow rate' response during induced bronchoconstriction is a decrease in indices of maximal flow and has been considered by some to indicate peripheral bronchoconstriction while a decrease in specific airway conductance ('conductance response') has been considered to indicate central bronchoconstriction (Bouhuys and van de Woestijne, 1970). While it is true that in normal man a large portion of R°w is in the upper airways (Fig. 3), the relative contribution of these airways to Row decreases in the presence of peripheral (or generalized) bronchoconstriction. Authors have been unable to confirm the distinction between a flow rate and a conductance response and have found simultaneous decreases in conductance and maximal flow rates in the same subjects after bronchial challenge (Olive and Hyatt, 1972; Mansell et al., 1974). That it is unjustified to equate reduction in l?max with peripheral bronchoconstriction and that the same maximal flow can result from events in both peripheral and central airways is suggested by the finding of Antic and Macklem (1976) that bronchodilator treatment of asthmatic subjects increased their helium response but left MEFV curves while breathing air unchanged. To explain this they assumed that the same decrease in smooth muscle tone lowered peripheral resistance but made central, flow limiting airways more collapsible. 1.5.3.6. Other methods to produce or assess selective bronchoconstriction. Jackson et al. (1977) applied an acoustic pulse with high energy content at frequencies between 250 and 10,000 Hz (i.e. wavelengths comparable to linear airway dimensions) to dog lungs, and measured the amplitude of the pressure waves reflected at different times after pulse generation with a microphone. The amplitude gave information about total cross-sectional area, and the travel time of the pulse gave information about the distance down the bronchial tree at which a particular cross section was measured. An area-distance plot was thus obtained, which gave an indication of the longitudinal distribution of cross-sectional areas of the airways. After bronchoconstriction induced by carbachol or by histamine, a strong correlation was found between area changes measured on tantalum bronchograms at various distances down the airways and the measurements obtained by the acoustical method. Lynn-James (1969) used an in vivo tracheal preparation of the guinea pig to assess 'large' airway responses to drugs and used a Konzett-R6ssler preparation as an indicator of'small airway' responses. Yeates et al. (1976) and Ruffin et al. (1978) achieved 'central' deposition of aerosolized histamine in the trachea and main bronchi by injecting the aerosol as a bolus at the end of a normal inspiration. Inhaling histamine as a fine aerosol (particle size, 1.5 #m) with a slow, full breath from FRC to TLC and then breath holding for 20 sec caused diffuse deposition as confirmed by radiotracers. 1.5.4. Influence o f prior tone on subsequent bronchoconstriction Prior airway calibre may influence subsequently induced bronchoconstriction at various levels. (a) In vitro studies show that the active tension developed by smooth muscle during electrical stimulation depends on the initial length of the muscle (Stephens et al., 1969). It is possible that an increase in resting tone (e.g. in an asthmatic subject) puts the muscle at a more or less advantageous length for the development of subsequent tension. (b) If this variable is eliminated by stretching a muscle to its optimal length, (leading to maximum tension development following stimulation) usually finds a sigmoid shape of the dose-

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FIG. 15. ‘Magnification’ of small differences in airway calibre by the resistance measurement which varies with the fourth power of the radius. When two airways of slightly differing radius (1.0 vs 0.9, arbitrary units, left side)are constricted by the same amount (0.2 mm, right side), this will lead to a disproportionate increase in the resistanceOf the airway that was slightly narrower initially.

response curve to a bronchoactive drug. Prior bronchoconstriction may be like operating on the steeper portion of the curve, where the same stimulus will evoke a much greater response (Benson and Graf, 1977). (c) If resistance is the variable measured in duo, the fourth power relationship between the radius of an airway and its resistance may magnify these differences (Fig. 15) (Benson, 1975). (d) Folding of the mucosa with bronchoconstriction, smooth muscle hypertrophy or mucosal oedema will further add to the magnification (Hutt and Wick, 1956 ; Bouhuys, 1963; Freedman, 1972). Thus, geometrical factors may complicate the interpretation of experiments designed to study reflexes and the interaction of bronchoactive drugs. Nevertheless, specific experiments can be devised which eliminate geometrical factors from the interpretation. The following are examples of studies that eliminated successfully the possibility that effects were due to differences in prior tone. (a) Green and Widdicombe (1966) were able to reduce CO,-induced bronchoconstriction in dogs by vagotomy but vagal section also reduced baseline tone. Since restoration of tone by electrical stimulation of the vagi did not restore the CO2 response, differences in baseline tone could not explain the effect of vagotomy, and they concluded that the effect of CO1 was reflex. The same method was used by DeKock et al. (1966). (b) Serotonin caused much greater bronchoconstriction before than after cooling both cervical vagi in dogs (Hahn et al., 1978). This could not be attributed to differences in basal airway tone for two reasons : first, similar amounts of bronchoconstriction induced by acetylcholine or histamine were little or not at all affected by vagal blockade; and second, when basal tone was increased by serotonin and acetylcholine in vagotomized dogs, only serotonin but not acetylcholine increased the effect of subsequent vagal stimulation (Fig. 16). The authors concluded that serotonin and the vagi interacted in producing bronchoconstriction. (c) In a number of studies involving bronchial challenge in normal and asthmatic subjects, large differences in bronchomotor responses to inhaled bronchoactive drugs were found, although baseline resistance values were the same (Cade and Pain, 1971; Rubinfeld and Pain, 1977 ; Empey et al., 1976; Orehek et al., 1977; Golden et al., 1978). 1.55. ReJex bronchconstriction Afferent pathways may arise from intrapulmonary or extrapulmonary sensory receptors (Fig. 17A, B), the efferent pathway is in the vagus nerve. Drug-induced reflex bronchoconstriction has to be distinguished from direct effects of the drug or smooth muscle (Fig. 17C) and from interaction between the efferent vagus nerve and the drug being studied (Fig. 17D, Fig. 16). The following methods can be used to prove reflex bronchoconstriction. 1.5.5.1. Efferent or combined uflerent and ej%rent ougul blockade. Abolition of drug effects by vagotomy, by complete cold block of the vagi or by atropine will only help to exclude direct smooth muscle effects but does not provide sufficient evidence for a reflex. However, the same procedures will be perfectly adequate if the stimulus is limited to extrapulmonary receptors

276

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FIG. 16. Effect of repetitive stimulation of the peripheral ends of both cut cervical vagus nerves on total pulmonary resistance under control conditions and during application of aerosols of acetylcholine (0.03 per cent) and serotonin (0.2 per cent). Each arrow represents one vagal stimulus. Period of drug application indicated at top of each panel.

and only the efferent limb of the reflex arc is in the vagus nerve as in bronchoconstriction caused by laryngeal irritation (Kahn, 1907; Nadel and Widdicombe, 1962a). 1.5.5.2. Differential blockade of the vagus nerves. This is blockade of afferent but not efferent fibres in the nerves. However, none of the procedures used to achieve differential blockade, e.g. differential cooling (Franz and Iggo, 1968), anodal hyperpolarization (Paintal, 1973) and application of local anaesthetics to the outside of the nerve trunk (Franz and Perry, 1974) or to the bronchial mucosa (Dain et al., 1975) have proved to be entirely selective for one fiber

A

B

C

D

Flo. 17. Possible modes of action of an agent (AgO which, when applied to only one lung, leads to bronchoconstriction. It can have a direct effect on smooth muscle (C) or can interact with local cholinergic mechanisms (D). It can also cause reflex bronchoconstriction by stimulating intrapulmonary (A) or extrapulmonary (B) sensory receptors (the latter being reached via the blood stream). Common to (A) and (B) is bilateral bronchoconstriction despite unilateral application of the agent, but there will be differences in the effect of unilateral vagal blockade: this will relieve constriction on both sides in A, but only on one side in B. Common to situations (C) and (D) is unilateral bronchoconstriction. Unilateral vagal blockade will only relieve bronchoconstriction in D.

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type and thus these procedures provide only suggestive evidence. Recently it has become possible to achieve almost complete blockade of pulmonary stretch receptors in rabbits by giving them 200 ppm SO2 to breathe for 5-10 min (Widdicombe, 1977). The effect is species dependent and has not been demonstrable in dogs. 1.5.5.3. 'Separation' experiments. Definitive proof of reflex bronchoconstriction has come from experimental preparations in which the organ containing the receptors is separated from the effector organ. (a) Divided lung experiments : Gold et al. (1972), using a double lumen catheter, applied aerosolized antigen to only one lung of allergic dogs. Bronchoconstriction occurred not only in the lung receiving the antigen but in the opposite lung as well. Cooling only the vagus nerve which innervated the lung receiving the antigen abolished the airway reaction in both lungs, proving a reflex arising from sensory receptors in the lung receiving the antigen (Fig. 17A). A divided lung preparation was also used by Ingrain (1975) to prove the reflex character of bronchconstriction induced by hypercapnia. (b) Separation of upper and lower airways has also been used to limit agents or stimuli to one segment of the airways while effects were studied in the other. SO2 delivered only to the trachea caused an increase in the resistance of the lower airways which could be prevented by vagal cooling or by atropine (Nadel et al., 1965). Conversely, large inflations of the lungs caused dilation of an isolated segment of trachea which could be blocked by cutting the pulmonary vagi or by differential cooling of the vagosympathetic nerves, procedures that left the efferent innervation of the tracheal segment intact (Widdicombe and Nadel, 1963). In this case the tracheal segment was the effector organ. The isolated tracheal segment has also been used to study reflex effects ofhypercapnia and hypoxaemia (Kahn, 1907 ; Loofbourrow et al., 1957 ; Nadel and Widdicombe, 1962b; Green and Widdicombe, 1966). Segments have been created surgically for chronic experiments and to study reflexes in awake animals (Graf et al., 1975). Points to be considered in this preparation are: first, the smooth muscle tone of the segment depends critically on the level of ventilation. No dilation will be demonstrable in a segment which is maximally dilated because of hyperventilation of the animal ; similarly, constriction will not be demonstrable in a segment already constricted maximally by hypoventilation. Second, drugs applied to the lungs may reach the segment via the blood stream and constriction of the segment will not prove a reflex. Additional evidence, e.g. results of nerve blockade or studies of neurone discharge (Stransky et al., 1973) is required. (c) Separation of circulations : one can restrict a stimulus to the distribution of one portion of the circulation while recording effects from parts of the lung perfused by another portion of the circulation. DeKock et al. (1966) restricted the delivery of histamine to the bronchial circulation by direct catheterization of the bronchial artery or by using special aortic catheters, and they recorded effects from a tracheal segment not perfused by the bronchial. artery. Restriction of delivered materials to the pulmonary circulation is possible by occluding a pulmonary artery with a balloon catheter and injecting drugs distal to the balloon, using injection volumes that do not appear in the systemic circulation. Effects of pulmonary artery occlusion itself (airway smooth muscle contractions, changes in local blood gas tensions) have to be taken into consideration. 1.5.6. Hyperirritability Hyperirritability, a greater than normal smooth muscle reaction to bronchoconstrictor drugs, is not restricted to clinical or experimentally induced asthma, which we will not consider here, but is a more general phenomenon. It occurs transiently in otherwise normal subjects during viral infections (Empey et al., 1976), after administration of live attenuated influenza virus (Laitinen et al., 1976a) and can be produced experimentally in dogs and in man by exposure to ozone (Lee et al., 1977 ; Golden et al., 1978). Cholinergic mechanisms must be involved because the increase in the response to histamine in these conditions can be prevented by atropine or by cold block of the vagi. Reflex bronchoconstriction may be the cause, but local effects are also likely. For example, Murtagh et al. (1971) found increased

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reactivity to mecholyl in lungs excised from dogs with kennel cough, suggesting that local mechanisms contribute. One practical consequence should be to attempt to eliminate infection when studying smooth muscle physiology. 1.5.7. Hypoxia and hypercapnia 1.5.7.1. Reflex and direct effects on smooth muscle. Arterial hypercapnia causes reflex bronchoconstriction (Roy and Brown, 1885; Dixon and'Brodie, 1903; Loofbourrow et al., 1957; Nadel and Widdicombe, 1962b; Green and Widdicombe, 1966; Ingram, 1975). It is not mediated by the chemoreceptors as are similar but weaker effects of hypoxia (Nadel and Widdicombe, 1962b), but may be due to hypercapnia of the CNS as was shown in crossedcirculation experiments (DeBurgh-Daly et al., 1953). In two studies in man, increased Raw during hypercapnia was not prevented by atropine and was thought to be due to laryngeal constriction (Sterling, 1969; Rodarte and Hyatt, 1973). Isocapnic hypoxia caused very small decreases in specific airway conductance in man (Saunders et al., 1977). Alveolar hypocapnia, which has been achieved experimentally by hyperventilation (Newhouse et al., 1964; Sterling, 1968) or by temporary occlusion of one pulmonary artery, producing CO 2 levels close to zero (Severinghaus et al., 1961 ; Swenson et al., 1961 ; Ingram, 1975) caused an increase in airflow resistance, partially reversible by atropine (Newhouse et al., 1964; Sterling, 1968; Ingram, 1975) and, at least during pulmonary artery occlusion, a decrease in compliance, which is unaffected by vagotomy (Severinghaus et al., 1961 ; Ingram, 1975), suggesting a direct smooth muscle effect on peripheral airways, reversible by CO 2 and probably mediated by changes in pH (Coon et al., 1975). The bronchoconstrictive effects of arterial hypercapnia and alveolar hypocapnia are additive (Ingram, 1975). Breathing CO2 relieved exercise-induced branchoconstriction in asthmatic patients (Fisher et al., 1970), even if arterial pCO2 was normal (Fisher and Hansen, 1976). Perhaps this relates to earlier findings by Nisell (1950) that CO2 relaxed isolated perfused cat lungs contracted by carbachol and by Wick (1952) that CO2 relaxed isolated tracheal rings contracted by pilocarpine. This leads to the second important aspect of the effects of CO2. 1.5.7.2. Interactions between C02 and bronchoactive drugs. In anaesthetized dogs, Sterling et al. (1972) studied the effect of inhaling CO2 on bronchoconstriction produced by stimulation of the vagi or by infusion of acetylcholine or serotonin: CO 2 inhibited only the increase in RL due to serotonin and this was probably due to acidosis because it could be reproduced by infusion of HCI. Similarly, in isolated bronchial rings 10 per cent CO 2 or a decrease in pH inhibited only serotonin- but not acetylcholine-induced constriction. Observations were extended by Duckies et al. (1974) who studied the relaxing effects on contractions produced by methacholine, bethanechol and carbachol in cats. 1.5.8. Effects of anaesthesia Dixon and Brodie (1903) noted in cats that, after anaesthesia with urethane, injections of muscarine no longer produced bronchoconstriction, that the inhalation of ether or chloroform abolished the effect of the vagus on the bronchioles and that all three anaesthetics induced dilation of the bronchi when constriction was present. Very deep anaesthesia, no matter how achieved, will abolish reflexes and prevent airway constriction irrespective of the drug given. But even careful, light anaesthesia will have effects. Urethane inhibits histamine-induced bronchoconstriction in rabbits (Douglas et al., 1972a). Baseline RL was lower during halothane than during cyclopropane anaesthesia in dogs and both anaesthetics inhibited histamine-induced bronchoconstriction as confirmed by tantalum bronchography (Hickey et al., 1969). Lv. barbiturates can cause bronchoconstriction and dilation in dogs and can block the effects of vagal stimulation (Bernstine et al., 1957). Catecholamines are released by urethane and, to a smaller degree barbiturates in rats (Spriggs, 1965), cyclopropane in man (Price et al., 1959 ; Millar and Morris, 1961) but not in dogs (Millar and M orris, 1961), and by ether-oxygen, divinyl-ether and chloroform in dogs

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(Millar and Morris, 1961; Richardson et al., 1957). Changes in acid-base balance and oxygenation during light chloralose and pentobarbitone anaesthesia have been followed in dogs for 8 hr (Ledsome et al., 1971). At 30 min pentobarbitone reproducibly caused hypercapnia and acidaemia which later on subsided. The persistent non-respiratory acidaemia (average pH 7.25, pC0, = 48.3 mm Hg) seen during chloralose anaesthesia was attributed to the unbuffered solvent used. Obviously such effects are important in view of the influence of COZ and pH on airway tone and on the action of bronchoactive drugs. Regular blood gas measurements are indispensable. The increase in RL and the decrease in CL occurring in artificially ventilated animals can be prevented by repeated full inflation of the lungs (Mead and Collier, 1959; Douglas et al., 1974). Increases in R,, following administration of curare (attributed to histamine release) have been reported by a number of authors (e.g. Crago et al., 1972), but have not always been confirmed (Gerbershagen and Bergman, 1967; Kilburn, 1960). Anaesthesia is critical in the study of reflex effects. Depending on receptor type and concentration of the anaesthetic, several volatile anaesthetics can either increase or depress the afferent activity of sensory nerve endings (Coleridge et al., 1968; Younes and Youssef, 1978). 1.59. Species diferences Vagal stimulation appears to constrict mainly peripheral airways in the sheep, causing a fall in compliance and airway closure (Colebatch and Halmagyi, 1963) whereas in most other species it constricts predominantly central airways (Olsen et al., 1965 ; Karczewski and Widdicombe, 1969 ; Clarke et al., 1970) and will not lead to airway closure (Woolcock et al., 1969a, 1969b). Rhesus monkeys have much lower lung elastic recoil than most other species (Part et al., 1978) and there are species differences in the shape of the MEFV-curve and the location of the equal pressure point (Macklem and Mead, 1968). Excised cat lungs can be relaxed with isoproterenol, i.e. seem to have intrinsic tone, but dog lungs cannot (Colebatch and Mitchell, 1971). These are but a few examples.

2. METHODS

TO STUDY AIRWAY SMOOTH

MUSCLE IN VITRO

2.1. ISOLATED WHOLE ORGANS 2.1.1. Isolated lungs and isolated perfused lungs Methods are essentially the same as in uivo and some of the studies already mentioned were performed on excised lungs. Not all in viuo techniques can be used, e.g. plethysmography for R,, measurements would be difficult. Oscillation techniques, on the other hand, are very easy to perform in lungs distended either by negative pressure (in an evacuated container) or by positive pressure inflation through the loudspeaker system (e.g. Macklem and Mead, 1967). Excellent tantalum bronchograms are obtained in excised lungs (Nadel et al., 1968; Hughes et al., 1972, 1975) or trachea (Jones et al., 1975a, 1975b) and air bronchograms give more detail in excised lungs than in situ (Murtagh et al., 1971). The following problems may be encountered by those working with excised lungs (a) Compliance curves differ slightly from those of lungs in viuo and RV is higher (Wohl et al., 1968 ; Pare et al., 1978); air trapping occurs early, sometimes after a few inflations (Faridy and Permutt, 1971) but can be reversed by degassing lungs in a vacuum jar. (b) Excised lungs will stiffen during artificial ventilation, especially when they are hyperinflated repeatedly. This is due to inactivation of surface active material and can be minimised by avoiding anoxia, maintaining a positive end-expiratory pressure, using negative instead of positive pressure ventilation and keeping temperature at 37°C. (c) Although vagotomy removes all airway tone in uioo, it cannot be assumed that excised lungs have no smooth muscle tone, because tone has been demonstrated in isolated cat (Dixon and Brodie, 1903 ; Colebatch and Mitchell, 1971) and dog (Goldman and Puy, 1975) lungs. Its cause is obscure. The fact that smooth muscle tone induced post mortem by

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histamine can be abolished by EDTA (Clay et al., 1977) speaks against rigor as its cause (Bose, 1976). (d) Isolated perfused lungs have the additional problem of pulmonary oedema formation and this can also happen in vivo (Sladen et al., 1968). They should not be perfused with blood because of the ensuing mediator release ('bronchotonins'--Nisell, 1950; Gaddum et al., 1953), leading to severe bronchoconstriction. Careful handling of the preparation will lessen this somewhat (Coon et al., 1975). Even so, the interpretation of smooth muscle experiments in isolated, bloodperfused preparations is difficult. 2.1.2. The isolated whole bronchial tree By dissecting away all the parenchyma and by comparing bronchograms of the isolated bronchial tree to those obtained in the intact lung, Hyatt and Flath (1966) showed that the lung parenchyma had little influence on airway dimensions. Iravani et al. (1971) dissected the whole bronchial tree of rats, put it in a shallow water bath at 37°C and, using a stereomicroscope and an image splitter, traced it before and after a variety of interventions, airway pressure being maintained at 3-5 cm H20. Constrictor agents affected proximal more than distal bronchi. Even when they had been constricted by acetylcholine, 10-80 per cent CO2 did not dilate bronchi. Since these results are in contrast to those of other investigators who studied airway dimensions in intact lungs, perhaps the lung parenchyma in some way influences the bronchomotor response ofintrapulmonary airways to CO2 or drugs (see also Section 1.5.4). Others have placed thin lung slices (obtained by sectioning gelatinfilled lobes) in a saline-filled Petri dish under a microscope to observe the reaction of large and small bronchi to drugs that were added to the bath (Sollmann and Gilbert, 1937). 2.1.3. The isolated tracheal tube This has been used as a pharmacological preparation, when the variable measured was either the volume of air displaced (isotonic contraction. Jamieson, 1962; Carlyle, 1964) or the increase in pressure of the liquid-filled tube (isometric contraction, Guirgis, 1969 ; Farmer and Coleman, 1970). Carlyle (1964) also assayed acetylcholine released into the tracheal lumen or into the organ bath during transmural electrical stimulation. Comparison with a pure muscle preparation (trachealis) showed that most of the acetylcholine released by the tracheal tube came from muscle. Others have used the isolated trachea for mechanical studies, showing its compressibility was greater when smooth muscle contraction was absent. Variables measured were either the resistance to air flow (Bryant et al., 1970; Coburn et al., 1972), pressure-flow-volume or pressure-flow-area characteristics during maximal flow (Knudson and Knudson, 1973, 1975 ; Jones et al., 1975a, 1975b) or the pressure-volume behaviour when there was no flow (Olsen et al., 1967b, 1967c; Coburn et al., 1972). Olsen et al. made similar measurements on bronchial tubes. H~tkansson and Toremalm (1967) measured the longitudinal tension of rabbit tracheal tubes, demonstrating spontaneous rhythmic fluctuations in tension accompanied by fluctuations in membrane potential. 2.2. THE ORGAN BATH 2.2.1. Tracheal and bronchial chain preparations These are among the most frequently used preparations. Castillo and DeBeer (1947) sectioned the guinea pig trachea into 12 rings, tied them together with their muscle strips in alignment and mounted them in a bath. No weights had to be applied (except to keep the chain vertical) because the cartilage with its tendency to spring open kept the muscle under tension. Atropine did not produce relaxation, suggesting there was no spontaneous release of acetylcholine post mortem. Bronchial chains from an asthmatic patient contracted and released histamine upon addition of specific antigen to the bath (Schild et ai., 1951). Electrical field stimulation in the presence of adrenergic and cholinergic blockade (using propanalol and atropine) caused relaxation of guinea pig tracheal chains and this was more

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pronounced after prior constriction with histamine or (in chains from sensitized animals) with specific antigen (Richardson and Bouchard, 1975). Since the relaxation could be blocked by tetrodotoxin, suggesting it was neuronally mediated, the authors interpreted their findings as evidence for a nonadrenergic inhibitory nervous system in the trachea, confirming earlier studies by Coburn and Tomita (1973) in trachealis muscle and by Coleman and Levy (1974) in isolated tracheal tubes. Bronchial and tracheal chains from several species were also used by McDougal and West (1953) who modified the technique somewhat by cutting the cartilage in each ring (without opening it). To obtain greater sensitivity Ak~asu (1952) opened the rings by cutting through the cartilage. Later he also cut away most of the cartilage (1959) and tied the remaining cartilaginous ends together. Like McDougal and West (1953) he found considerable species differences in the reaction to histamine and acetylcholine. Foster (1960) modified the technique further by connecting the odd numbered (opened) rings from one animal with the even numbered ones from the other, minimising variability due to inter-individual differences in sensitivity. Using this technique, Carlyle (1963) confirmed the absence of spontaneous acetylcholine release from guinea pig trachea and Foster (1966) found no evidence for the presence ofalpha-adrenergic recePtors in guinea pigs. 2.2.2. Tracheal and bronchial spirals Rosa and McDowell (1951), working with human bronchi, compared the bronchial chain preparation with a preparation in which bronchi were cut spirally and were suspended in the bath as one piece of tissue. Less manipulation was involved and the preparation was more sensitive than the chain, which in turn was more suitable when bronchi were too short to yield a spiral of sufficient length. Addition of flour-dust extract to the bath produced a marked and lasting constriction (accompanied by histamine release) only in the preparation 4"rom an asthmatic miller with a positive skin reaction to flour-dust but not in preparations from normal subjects. Patterson (1958) obtained similar reactions on addition of antigen to spirals from sensitized guinea pigs. The method was rediscovered by Constantine (1965) and is still widely used : Fleisch et al. (1970), contrary to Foster (1966) found evidence for alphaadrenergic receptors in spirals from several species. Orehek et al. (1975a) showed that several smooth muscle constricting agents caused prostaglandin release from guinea pig tracheal spirals and that prostaglandins in turn modulated the contractions. 2.2.3. Tracheal and bronchial rings Single tracheal (Danko et al., 1968) and bronchial (Stephens et al., 1968) rings instead of multiple ring preparations have come into use more recently. Tension is usually recorded isometrically. Preparations are used for pharmacological (Sterling et al., 1972; Duckies et al., 1974) and physiological studies. Stephens et al. (1968) used this preparation to construct length-tension and stimulus-response curves for bronchial smooth muscle at various levels of pH, pO2 and pCO2. Similar studies have been performed on short segments (4-5 rings long) of trachea mounted like a single ring (i.e. with the axis of the trachealis muscle vertical (Souhrada and Dickey, 1976). Hooker et al. (1977) described a cheap and easy modification, using two 30-gauge disposable needles bent into shape to mount bronchial rings of very small diameter (about 1 mm) for isometric recording. 2.2.4. Tracheal and bronchial muscle strips Tendelenburg (1912) and Macht and Ting (1921) obtained bronchi of 10 mm diameter from calves and pigs, cut these into rings 5 mm wide, opened the rings, removed all cartilage and mounted the remaining piece of tissue containing muscle and mucosa in a bath. Gaddum and Stephenson (1958) recorded contractions of the muscle from a single ring of guinea pig trachea in a special microbath containing only 5 ml fluid. A 'piece of muscle removed from the inside of one ring of the trachea' has been used extensively in studies of adrenergic and serotonin receptors (e.g. Offermeier and Ariens, 1966). More recently, Stephens and his group

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systematically applied methods developed to study the mechanics of striated muscle to the canine trachealis muscle (which connects the posterior ends of each ring of trachea), because it can be separated from the mucosa easily, is the 'purest' muscle preparation of the bronchial tree (75 per cent muscle, Stephens et al., 1969) and has other convenient properties: it has parallel fibres, little resting tension, no spontaneous activity, and it can be tetanized. Studies of airway smooth muscle mechanics have been reviewed recently (Stephens, 1975). Trachealis muscle is used increasingly for various applications: in field stimulation studies Coburn and Tomita (1973) obtained pharmacological evidence for the existence of a nonadrenergic inhibitory nervous system in guinea pigs. Trachealis muscle was used in a study of smooth muscle rigor due to metabolic inhibition (Bose, 1976), in electrophysiological studies (Kirkpatrick, 1975 ; Coburn and Yamaguchi, 1977 ; Cameron and Kirkpatrick, 1977) and in investigations combining electrophysiological and tension recordings with assays of high energy phosphates Bose and Bose, 1977) or prostaglandins (Yamaguchi et al., 1976). 2.2.5. Tissue strips Kapanci et al. (1974), and more recently Lulich et al. (1976) and Gryglewski et al. (1976), have used strips of lung parenchyma to record tension changes in or mediator release from the most peripheral parts of the lung. Lulich et al. elicited strong contractions in both lung strips and tracheal chains by applying Ascaris antigen to preparations from actively sensitized cats. They also found differences between trachea and tissue strips in the response to several mediators as did Drazen and Schneider (1977). 2.3. SUPERFUSION METHODS

The principle of running fluid containing an active substance over the surface of a tissue suspended in air (instead of placing it in a bath) was first described for rabbit intestine by Finkleman (1930) and his was a nerve-muscle preparation comparable to the one used by Bouhuys (1974) (Fig. 18). Drugs were first injected into the stream of fluid running over the tissue, but Gaddum (1953) obtained greater sensitivity by stopping the flow for a standard

Tyrode Tyrode

t

-30Hz

A

i¸'¸

L

. drain

FIG. 18. Superfused guinea pig tracheal tube with vagus nerve attached (from Bouhuys, 1974).

Methods of study of airway smooth muscle and its physiology

283

interval and applying the active solutions undiluted to the surface of the muscle. Both methods are frequently used in a variety of assays (Stascewska-Barczak and Vane, 1965; Orehek et al., 1975a; Tiirker and Zengil, 1976) and often several tissues are superfused in a row (cascade) to make assays more specific for certain mediators. Prostaglandins were released upon electrical or mechanical stimulation from superfused spirals (Orehek et al., 1975a) and chains (Tfirker and Zengil, 1976). A tracheal nerve-muscle preparation from the guinea pig has recently been developed (Fig. 18, from Bouhuys, 1974), which is used to assess the interaction between mediators and the efferent vagus nerve (Douglas et al., 1972b) and this technique might help to clarify the relative contribution of reflex, direct and interaction effects to the hyperreactivity of smooth muscle seen under various conditions. 2.4. ELECTROPHYSIOLOGICALMETHODS

Basically these involve intracellular (microelectrode) and extracellular (single or double sucrose gap) recordings. Since they are not specific for airway smooth muscle and have been reviewed recently (Coburn et al., 1975; Kuriyama and Ito, 1975), they will not be considered further. 2.5. SPECIAL PROBLEMS ENCOUNTERED IN STUDIES OF AIRWAY SMOOTH MUSCLE IN VITRO

2.5.1. Species and age differences Species differences have been mentioned (McDougal and West, 1953 ; Akeasu, 1959) and need no further emphasis. An age-related reduction in the sensitivity of rat and guinea pig tracheal chains to isoproterenol has been reported by Aberg and Adler (1973). 2.5.2. The importance of prior tone Ideally a muscle should be at L~x, the length at which subsequent stimulation produces the maximal response (Stephens et al., 1968, 1969). In experiments by Downes and Loehning (1977), the tone of a tracheal chain preparation before addition of local anaesthetics was crucial in determining whether contraction or relaxation was the dominant drug effect. In the lung strips of Lulich et al. (1976), the same dose ofisoproterenol caused a relaxation of 0.3 g when the initial tension was 1 g, but one of 2.3 g at an initial tension of 5 g. This agrees with findings in vivo, where only epinephrine and lung distension together, but not either procedure alone, completely relieved histamine-induced bronchoconstriction (Melville and Caplan, 1948). Not only does it matter whether or not there is tone; the exact cause of smooth muscle tone also makes a difference: whereas prostaglandin Ex had no effect on the intrinsic tone of the lung strip, it relaxed the carbachol-induced tone of trachea (Lulich et al., 1976). The degree of tone encountered may be related to the method of killing the animal. Exsanguination appears to be very effective in causing high resting tone (Trendelenburg, 1912; Macht and Ting, 1921). 2.5.3. Central vs peripheral airways Table 1 lists preparations (always taken from the same animal) used by investigators to assess the serial distribution of drug effects or of mediator release. Almost invariably differences were found between central and peripheral airways from the same animal and sometimes these differences were qualitative (e.g. Kapanci et al., 1974; Gryglewski et al., 1976). They might be related to differences in the direct action of drugs on smooth muscle or to differences in local indirect mechanisms, e.g. locally stored transmitter. Thus relaxations produced in guinea pig trachealis muscle by field stimulation were in part mediated by adrenergic mechanisms in the cervical portion of the trachea, but were almost entirely nonadrenergic in the thoracic portion, suggesting a decrease in adrenergic nerve endings towards the periphery in this species (Coburn and Tomita, 1973). J.P.T.7/2--F

H . L . HAHNand J. A. NADEL

284

TABLE1. In vitro Preparations of Airway Smooth Muscle Commonly Used to Differentiate Drug Effects on Central and Peripheral Airways Species Cat Guinea pig Sheep Rabbit Rats

Several Several (including man) Man

Central airways Tracheal chains Isolated tracheal tube (in vitro) Bronchial chains (major bronchi) Tracheal spirals

Peripheral airways Konzett-R/Sssler (in vivo) Konzett-RSssler (in vivo)

Main, 1964 Guirgis, 1969

Bronchial chains (less than 4 ram) Bronchiolar spirals

Eyre, 1969

Whole bronchial tree dissected free and observed under microscope. Bronchial spirals Parenchymal strips Spirals from 3-6 mm bronchi

Rings from 1 mm bronchioli

Tracheal and bronchial muscle strip Tracheal chains

Bronchial and bronchiolar rings Parenchymal strips

Guinea pig

Bronchial spirals (proximal bronchi) Tracheal spirals

Bronchial spirals (distal bronchi) Parenchymal strips

Guinea pig

Tracheal spirals

Parenchymal strips

Cat Man

Reference

Somlyo and Somlyo, 1970 Iravani et al., 1971 Kapanci et al., 1974 Persson and Ekman, 1976 Richardson and B61and, 1976 Lulich et al., 1976 Lo et al., 1976 Gryglewski et al., 1976 Drazen and Schneider, 1977

2.5.4. The possible importance of attached tissue Hypoxia is known to cause pulmonary vasoconstriction but does not constrict isolated pulmonary blood vessels (Lloyd, 1968). Mediators released from the surrounding parenchyma are believed to cause hypoxic vasoconstriction. That lung tissue attached to bronchial preparations may likewise contribute to their reactions is suggested by certain observations: (a) Orehek et al. (1975a) noted prostaglandin release from tracheal spirals upon scratching the tracheal mucosa and this might explain the discrepancy between several reports of spontaneous tracheal activity in vivo and in vitro (Loofbourrow et al., 1957; H~tkansson and Toremalm, 1967; Souhrada and Dickey, 1976) and the reported perfect quiescence of the trachealis muscle preparation (Stephens et al., 1969). (b) Lung parenchymal strips contracted with hypoxia and epinephrine whereas bronchial strips relaxed with both (Kapanci et al., 1974). The authors thought the parenchymal reactions were caused by tissue other than bronchial musculature. Since they discovered contractile fibrils in alveolar interstitial cells they speculated that these might be the other tissue. Mediator release from the surrounding tissue would be an alternative explanation and contributions from vascular smooth muscle a third. (c) Since it is more difficult to free small bronchial rings from attached tissue than large ones before mounting in a bath (the procedure is in any case incomplete), differences in amount of tissue attached might explain some of the discrepancies found between large and small airway preparations or the results reported by different investigators. II. FUNCTION OF AIRWAY SMOOTH MUSCLE Airway smooth muscle extends from the trachea to alveolar ducts (Nadel et al., 1964; Colebatch et ai., 1966; Clarke et al., 1970). Maybe even alveolar interstitial cells contain contractile elements (Kapanci et al., 1974), but if they do, they react differently from airway smooth muscle. Alveolar duct constriction has not been demonstrated by direct anatomical methods in man as it has been in animals but decreases in compliance following intravenous histamine (Laitinen et al., 1976b) suggest that in principle peripheral airways can constrict in man, even though this may be masked by catecholamine release (Ploy-Sang-Song et al., 1978) or by a volume history involving full lung inflation.

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Innervation of airway smooth muscle is predominantly cholinergic as evidenced by anatomical (Hebb, 1969; Fillenz, 1970; Mann, 1971) and physiological (Nadel et al., 1971 ; Woolcock et al., 1969a, b) studies but a weak adrenergic dilator influence also exists as has been shown by sympathetic nerve stimulation in the presence of smooth muscle tone (Cabezas et al., 1971) or by the increased effectiveness of vagal stimulation in the presence of adrenergic blockade (Woolcock et al., 1969b). Histochemical studies confirm the presence of adrenergic endings in bronchial smooth muscle. The receptors are predominantly betaadrenergic causing bronchodilation when stimulated. Alpha-adrenergic receptors seem to be few in normal airways (Fleisch et al., 1970; Himori and Taira, 1976) and could not be demonstrated in some studies (Foster, 1966; Danko et al., 1968). Cholinergic and adrenergic innervation extends to airways of the order of 1 mm in the dog but become less effective in smaller airways as was shown in nerve stimulation experiments (Olsen et al., 1965 ; Nadel et al., 1971 ; Cabezas et al., 1971). The presence of a third, 'nonadrenergic' inhibitory nervous system has been postulated in pharmacological studies (Coburn and Tomita, 1973 ; Coleman and Levy, 1974; Richardson and Bouchard, 1975 ; Richardson and Beland, 1976), but needs morphological confirmation. Airway calibre can be altered by a number of reflexes. Airway constriction during hypoxia, hypercapnia and laryngeal irritation originates from extrapulmonary sensory endings, and the efferent limb is in the vagus nerve. In other reflexes the vagus carries the afferent pathway as well. These include bronchoconstriction caused by inhalation of SO2 (Nadel et al., 1965), aerosols of dust, citric acid, histamine and cold air (Simonsson et al., 1967 ; DeKock et al., 1966), inhalation of antigen by sensitized dogs (Gold et al., 1972), the bronchoconstriction caused by rapid pulmonary deflation (Kahn, 1907; Widdicombe and Nadel, 1963a), or by injection of histamine into the bronchial artery (DeKock et al., 1966), and the bronchodilation caused by lung inflation (Kahn, 1907; Widdicombe and Nadel, 1963a). Bronchoconstriction due to hypocapnia (induced by pulmonary artery occlusion, for example) is a predominantly direct effect on the peripheral airways (Severinghaus et al., 1961 ; Ingram, 1975), causing a drop in compliance and a shift of ventilation to the opposite lung (Swenson et al., 1961). Pharmacological studies indicate that smooth muscle taken from different parts of the bronchial tree differs in intrinsic tone and in the reaction to a number of chemical agents. Reasons for this are not clear but may include mediator release from the surrounding tissue (Orehek et al., 1975a, b) and differences in the local distribution of nerve endings or transmitter (Coburn and Tomita, 1973). Whether this has relevance for human disease is not known. Some investigators maintain that sometimes predominantly central and sometimes predominantly peripheral airway constriction occurs in disease. However, this could also represent different stages of the same disease process, because in human studies 'central' and 'peripheral' reactors have always had disease of differing severity (Bouhuys et al., 1970; Mansell et al., 1974; McFadden et al., 1977). Compared with in vitro studies, in vivo assessment of central vs peripheral differences is further complicated by (a) reflex effects, (b) differences in distribution and metabolism of agonists due to route and mode of administration and (c) problems of measurement, because most of the methods used to assess airway size in vivo are weighted by the contribution of airways of certain sizes and are also influenced by variables other than airway size, especially elastic recoil. This makes it necessary to combine different measurements in order to locate the predominant site of constriction. What is the function of airway smooth muscle? Essentially we do not know. It has been suggested that airway smooth muscle is a phylogenetic remainder of a tissue important to lower animals, e.g. the lung fish which needs it to expel air against the water pressure (Bouhuys, 1974). Since airway calibre determines two conflicting variables, dead space and airway resistance, smooth muscle tone may help to maintain an optimal balance between the two (Widdicombe and Nadel, 1963b). Another suggestion has come out of the discovery that an increase in tone made trachea and large bronchi more resistant to compression. The same reflex stimulus that causes coughing may make airways less compressible. Against this is the finding of Jones et al. (1975b) that increases in recoil pressure above 20 cm abolished the

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increase in tracheal smooth muscle tone and maximal flow was indistinguishable from control. A third possible function is to make pulmonary ventilation more even and to match ventilation and perfusion. Thus, inhalation of carbachol caused a more uniform distribution of ventilation to the different parts of the lung in upright subjects (Engel et al., 1976) and in the human experiments of Swenson et al. (1961) ventilation shifted away from the lung with pulmonary artery occlusion. Mechanisms involved are not quite clear. Any interpretation in terms of resistance (RL) and compliance (CL) has to postulate an influence of smooth muscle tone on both Rr. and Ct, otherwise the time constant (RL x CL) would change and the system would become frequency dependent, i.e. the balance would be appropriate at one frequency but not another. Although pulmonary artery occlusion can cause a decrease in CL by hypocapnic constriction of peripheral airways it is doubtful whether similar events take place under more normal conditions. Certainly changes in vagal tone have had little influence on CL (Nadel et al., 1964; Karczewski and Widdicombe, 1969; Hahn et al., 1976). The greater uniformity of ventilation when tone is increased has also been explained by a greater range of pressures (including very low pressures) at which airways open during inflation (Macklem and Engel, 1975 ; Engel et al., 1976). Whether or not the contractile fibrils which perhaps exist in alveolar interstitial cells and which seem to be sensitive to hypoxia (Kapanci et al., 1974) have a function in ventilation/perfusion regulation as the authors postulate we do not know.

REFERENCES An~gG, G. and AOLEg, G. (1973) The effect of age on ~-adrenoceptor activity in tracheal smooth muscle. Br. J. Pharmac. 47: 181-182. AKASAKA,K., K o ~ o , K., ONO, Y., MUE, S., AnE, C., KUMAGA1,M. and ISE, T. (1975a) A new electrode for electromyographic study of bronchial smooth muscle. Tohoku J. exp. Med. 117: 49-54. AI~ASAKA,K., KosNo, K., ONO,Y., MUE, S., ABE,C., KUMAGAI,M. and ISE,T. (1975b) Electromyographic study of bronchial smooth muscle in bronchial asthma. Tohoku J. exp. Med. 117: 55-59. AKCASU,A. (1952)The actions of drugs on the isolated trachea. J. Pharm. Pharmac. 4: 671. AKCASU, A. (1959) The physiologic and pharmacologic characteristics of the tracheal muscle. Arch. int. pharmacodyn. ThOr. 122: 201-207. ALLANDER,C., INGELSTEDT,S., JONSON,B. and WESTLING,H. (1964) Constant volume flow. An aid in respiratory investigation. Med. exp. 11 : 253-258. AMDUR,M. O. and MEAD,J. (1958) Mechanics of respiration in unanesthetized guinea pigs. Am. J. Physiol. 192: 364-368. ANTIC, R. and MACKLEM,P. T. (1976) The influence of clinical factors on site of airway obstruction in asthma. Am. Rev. resp. Dis. 114: 851-859. ARONSSON, H., SOLYMAR, L., DEM~SEY, J., BJURE, J., OLSSON, T. and BAKE, B. (1977) A modified forced oscillation technique for measurements of respiratory resistance. J. appl. Physiol. Resp. Environ. Exercise Physiol. 42: 650-655. AVERY,W. G. and SACKNER,M. A. (1972) A rapid measurement of functional residual capacity in the paralyzed dog. J. appl. Physiol. 33: 515-518. BAR~,~ETT,T. B: (1967) Effects of helium and oxygen mixtures on pulmonary mechanics during airway constriction. J. appl. Physiol. 22: 707-713. BAgrER, C. E. and CAMPn~LL,A. H. (1973) Comparison of airways resistance measurements during panting and quiet breathing. Respiration 30:1-11. BARTLETT,R. G. JR., BRUBACH,H. F., TgIMBLE, R. C. and SPECHT, H. (1959) Airway resistance measurement during any breathing pattern in man. J. appl. Physiol. 14: 89-96. BASS, P. and WILEY, J. N. (1972) Contractile force transducer for recording muscle activity in unanesthetized animals. J. appl. Physiol. 32: 567-570. BAT'nSTA, S. P., STEnER, W. D., G~EN, M. and KENSLER,C. J. (1973) Technique for implanting lower airway catheters chronically in dogs. Arch. Environ. Health 27: 334-339. BENSON,M. K. (1975) Bronchial hyperreactivity. Br. J. Dis. Chest 69: 227-239. BENSON, M. K. and GgAF, P. D. (1977) Bronchial reactivity: interaction between vagal stimulation and inhaled histamine. J. appL Physiol. Resp. Environ. Exercise Physiol. 43: 643-647. BERGMAN, N. A. (1966) Measurement of respiratory resistance in anesthetized subjects. J. appl. Physiol. 21: 1913-1917. BERGMAN,N. A. and WALTEMATH,C. L. (1974) A comparison of some methods for measuring total respiratory resistance. J. appl. Physiol. 36: 131-134. BEgNSTI~,~,M. L., BEAKER,E. and CULLEN,M. (1957) The bronchomotor effects of certain intravenous barbiturates on vagal stimulation in dogs. Anaesthesiology 18: 866-870. BosE, D. (1976) Increase in resting tension of tracheal muscle due to rigor during metabolic inhibition. Am. J. Physiol. 231: 1470-1475.

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287

BosE, R. and BOSE, D. (1977) Excitation-contraction coupling in multiunit tracheal smooth muscle during metabolic depletion: induction of rhythmicity. Am. J. Physiol: Cell Physiol. 2:C8-C13. Boultuvs, A. (1963) Effect of posture in experimental asthma in man. Am. J. Med. 34: 470-476. Bounuvs, A. (1974) Bronchial asthma. In: Breathing, Physiology, Environment and Lung Disease, (ed.) A. BOUHUYS, Chapter 18, pp. 441-489. Grune & Stratton, New York & London. BOUHUYS, A., HUNT, V. R., KIM, B. M. and ZAPLETAL, A. (1969) Maximum expiratory flow rates in induced bronchostriction in man. J. clin. Invest. 48:1159-1168. BourluYs, A. and JONSON, B. (1967) Alveolar pressure, airflow rate, and lung inflation in man. J. appl. Physiol. 22: 1086-1100. BOUHUYS, A. and VAN DE WOmTIJNE, K. P. (1970) Respiratory mechanics and dust exposure in byssinosis. J. clin. Invest. 49: 106-118. BRlSCOE,W. A. and DUBOlS,A. B. (1958) The relationship between airway resistance, airway conductance and lung volume in subjects of different age and body size. J. clin. Invest. 37: 1279-1285. BRODY, M. J. and KADOWITZ, P. J. (1974) Prostaglandins as modulators of the autonomic nervous system. Fedn Proc. Fedn Am. Socs exp. Biol. 33: 48-60. BROWN, R., INGRAM,R. H. JR. and McFADDEN, E. R. JR. (1978) Effects of prostaglandin F2~ on lung mechanics in nonasthmatic and asthmatic subjects. J. appL PhysioL Resp. Environ. Exercise Physiol. 44: 150-155. BRYANT,G. H. and HANSEN,J. E. (1975) An improvement in whole body plethysmography. Am. Rev. resp. Dis. 112: 464-465. BRYANT, L. R., HOUCK, G. R., LOUGHRIN,J. R. and LIEBER,A. (1970) Tracheo-bronchial collapsibility. An in vivo and ex vivo study. Respiration 27: 74--85. BUTLER;J., C ~ o , C. G., ALCALA, R. and DuBols, A. B. (1960) Physiological factors affecting airway resistance in normal subjects and in patients with obstructive respiratory disease. J. clin. Invest. 39: 584-591. CAaEZAS, G. A., GRAF, P. D. and NADEL, J. A. (1971) Sympathetic versus parasympathetic nervous regulation of airways in dogs. J. appl. Physiol. 31: 651-655. CADE, J. F. and PAIN, M. C. F. (1971) Bronchial reactivity. Its measurement and clinical significance. Aust. N.Z.J. Med. 1: 22-25. CA~RO~, A. R. and KIRKPATRICK, C. T. (1977) A study of excitatory neuromuscular transmission in the bovine trachea. J. Physiol. 270: 733-745. CARLYLE, R. F. (1963) The mode of action of neostigmine and physostigmine on the guinea pig trachealis muscle. Br. J. Pharmac. 21: 137-149. CARLYLE, R. F. (1964) The responses of the guinea pig isolated intact trachea to transmural stimulation and the release of an acetylcholine-like substance under conditions of rest and stimulation. Br. J. Pharmac. 22: 126-136. CASTILLO,J. C. and HEBEER, E. J. (1947) The tracheal chain. 1. A preparation for the study of antispasmodics with particular reference to bronchodilator drugs. J. Pharmac. exp. Ther. 90: 104-109. C L ~ E , S. W., GRAF, P. D. and NAHEL, J. A. (1970) In vivo visualization of small-airway constriction after pulmonary microembolism in cats and dogs. J. appl. Physiol. 29: 646-650. CLAY, T. P., HUGHES,J. M. B. and JONES, H. A. (1977) Relationship between intrapulmonary airway diameter and smooth muscle tone in excised lungs. J. Physiol. 273: 355-365. CLEMENT, J. and VAN DE WO~ST1JNE, K. P. (1969) Pressure correction in volume and flow-displacement body plethysmography. J. appl. Physiol. 27: 895-897. CLEMENTS, J. A., SHARP, J. T., JOHNSOr~, R. P. and ELAM, J. O. (1959) Estimation of pulmonary resistance by repetitive interruption of airflow. J. clin. Invest. 38: 1262-1270. COBORN, R. F., OrmA, M. and TOM~TA,T. (1975) Recording of intracellular electrical activity with the voltageclamp technique with double sucrose-gap. In: Methods in Pharmacology, Volume 3, 'Smooth Muscle' pp. 247-260. (eds.) E. E. DANIEL and D. M. PATOU. Plenum Press, New York & London. COBURN, R. F., TnORr~TON, D. and ARTS, R. (1972) Effect of trachealis muscle contraction on tracheal resistance to airflow. J. appl. Physiol. 32: 397-403. COBORN, R. F. and TOMITA, T. (1973) Evidence for nonadrenergic inhibitory nerves in the guinea pig trachealis muscle. Am. J. Physiol. 224: 1072-1080. COBURN, R. F. and YAMAGUCHI,T. (1977) Membrane potential-dependent and -independent tension in the canine tracheal muscle. J. Pharmac. exp. Ther. 201: 276-284. COGSWELL, J. J. (1973) Forced oscillation technique for determination of resistance to breathing in children. Arch. Dis. Child. ,18: 259-266. COtEBATCH, H. J. H. and ENGELS, L. A. (1974) Measurement of lung volume in paralyzed cats. J. appl. PhysioL 36: 614-617. COLEBATCH, H. J. H. and ENGEL, L. A. (1974b) Constriction of the lung by histamine before and after adrenalectomy in cats. J. appl. Physiol. 37: 798-805. COLEBATCH,H. J. H., FINUCANE,K. E. and SM|TH, M. M. (1973) Pulmonary conductance and elastic recoil relationships in asthma and emphysema. J. appl. Physiol. 34: 143-153. COLEBATCIt, H. J. H. and HALMAGYI,D. F. J. (1963) Effect of vagotomy and vagal stimulation on lung mechanics and circulation. J. appl. Physiol. 18: 881-887. COLEBATCH, H. J. H., NAIL, B. S. and biG, C. K. Y. (1978) Computerized measurement of pulmonary conductance and elastic recoil. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44:611-618. COLEBATCH, H. J. H. and MITCHELL, C. A. (1971) Constriction of isolated living liquid-filled dog and cat lungs with histamine. J, appl. Physiol. 30: 691-702. COLEBATCH, H. J. H., OLSEN, C. R. and NADEL, J. A. (1966) Effect of histamine, serotonin, and acetylcholine on the peripheral airways. J. appL Physiol. 21: 217-226. COLEMAN,R. A. and LEvy, G. P. (1974) A non-adrenergic inhibitory nervous pathway in guinea pig trachea. Br. J. Pharmac. 52: 167-174. COLERIDGE, H. M., COLERIDGE,J. C. G., LUCK, J. C. and NORMAN,J. (1968) The effect of four volatile anaesthetic agents on the impulse activity of two types of pulmonary receptor. Br. J. Anaesth. 40: 484-492.

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H.L. HAHN and J. A. NADEL

COt.GAN, F. J. (1964) Relationship of changing bronchial caliber to respiration in the dog. J. appl. Physiol. 19: 803-811. COMROE, J. H. JR., BOTELHO, S. Y. and DuBols, A. B. (1959) Design of a body plethysmograph for studying cardiopulmonary physiology. J. appl. Physiol. 14: 439-444. COMROE, J. H., NISELL, O. I. and NIMS, R. G. (1954) A simple method for concurrent measurement of compliance and resistance to breathing in anesthetized animals and man. J. appl. Physiol. 7: 225-228. CONSTANTINE, J. W. (1965) The spirally cut tracheal strip preparation. J. Pharm. Pharmac. 17: 384-385. COON, R. L., RATTENBORG, C. C. and KAMPINE, J. P. (1975) Local control of pulmonary resistance and lung compliance in the canine lung. J. appl. Physiol. 39: 580-589. CRAGO, R. R., BRYAN,A. C., LAWS,A. K. and WINESTOCK,A. E. (1972) Respiratory flow resistance after curare and pancuronium, measured by forced oscillations. Can. Anaesth. Soc. J. 19: 607-614. CULVER, B., FRIEND,J., LOVERDE,V. and BUTLER,J. (1973) The flow limiting segment in the airway. Physiologist 16: 293. DAIN, D. S., BOUS,EV, H. A. and GOLD, W. M. (1975) Inhibition of respiratory reflexes by local anesthetic aerosols in dogs and rabbits. J. appl. Physiol. 38: 1045-1050. DAI~KO, G., TOZZl, S. and RUTH, F. E. (1968) Determination of the adrenergic mechanism of the isolated guinea pig trachea utilizing a new technique. Fedn Proc. Fedn Am. Socs exp. Biol. 27:351 (Abs. 772). DAWSON, S. V. and ELLIOTT, E. A. (1977) Wave-speed limitation on expiratory flow--a unifying concept. J. appL Physiol. Resp. Environ. Exercise Physiol. 43: 498-515. DEBURGH DALY, M., LAMBERTSEN,C. J. and SCHWEITZER,A. (1953) The effects upon the bronchial musculature of altering the oxygen and carbon dioxide tensions of the blood perfusing the brain. J. Physiol. 119: 292-341. DEKKER, E. and LEDEnOER, R. C. (1961) Compression of the tracheobronchial tree by the action of the voluntary respiratory musculature in normal individuals and in patients with asthma and emphysema. Am. J. Roentgenol. 85: 217-228. DEKOCK, M. A., NADEL, J. A., ZWI, S., COLEnATCH, H. J. H. and OLSEN, C. R. (1966) New method for perfusing bronchial arteries: histamine bronchoconstriction and apnea. J. appl. Physiol. 21: 185-194. DENNIS, M. W., DOUGLAS,J. S., CASBY,J. U., STOLWIJK,J. A. J. and BOUHUYS,A. (1969) On-line analog computer for dynamic lung compliance and pulmonary resistance. J. appl. Physiol. 26: 248-252. DESPAS, P. J., LEROUX, M. and MACKLEM, P. T. (1972) Site of airway obstruction in asthma as determined by measuring maximal expiratory flow breathing air and a helium-oxygen mixture. J. clin. Invest. 51 : 3235-3242. DIAMOND,L. (1967) Utilization of changes in pulmonary resistance for the evaluation ofbronchdilator drugs. Arch. int. pharmacodyn. Ther. 168: 239-250. DIAMOND,L. (1972) Potentiation ofbronchomotor responses by beta adrenergic antagonists. J. Pharmac. exp. ?'her. lgl: 434-445. DIRNAGL, K. (1953) Zur fortlaufenden Messung des Atemwiderstands. Z. Aerosol-Forsch. 2: 475-484. DIxoN, W. E. and BRODIE,T. G. (1903) Contributions to the physiology of the lungs. Part 1. the bronchial muscles, their innervation, and the action of drugs upon them. J. Physiol. 29: 97-173. DOLOVlCH, M. B., SANCtuS, J., ROSSMAN,C. and NEWHOUSE, M. T. (1976) Aerosol penetrance: a sensitive index of peripheral airways obstruction. J. appl. Physiol. 40: 468-471. DOSMAN,J., BODE, F., URBANETTI,J., MARTIN, R. and MACKLEM,P. T. (1975) The use of a helium-oxygen mixture during maximum expiratory flow to demonstrate obstruction in small airways in smokers. J. clin. Invest. 55: 1090-1099. DOUGLAS,F. G., CHONG,e. Y. and FINLAYSON, D. C. (1974) Effect of artificial ventilation on lung mechanics in dogs. J. appl. Physiol. 37: 324-328. DOUGLAS,J. S., DENNIS, M. W., RIDGWAY, P. and Bou,uYS, A. (1972) Airway dilation and constriction in spontaneously breathing guinea pigs. J. Pharmac. exp. Ther. 180: 98-109. DOUGLAS,J. S., HELGERSON, R. B. and BOUHUYS,A. (1972) Interaction of humoral agents and regulation of airway smooth muscle responses (ASMR). Fedn Proe. Fedn Am. Socs exp. Biol. 31:680 (Abs. 336). DOWNES, H. and LOEHNING, W. (1977) Local anesthetic contracture and relaxation of airway smooth muscle. Anesthesiology 47: 430-436. DRAZEN, J. M., LOmNG, S. H. and REGAN, R. (1976a) Validation of an automated determination of pulmonary resistance by electrical subtraction. J. appl. Physiol. 40:110-113. DRAZEN, J. M., LURING, S. H. and INGRAM,R. H. JR. (1976b) Localization of airway constriction using gases of varying density and viscosity. J. appl. Physiol. 41: 396-399. DRAZEN, J. M. and SCHNEIDER, M. W. (1977) Comparison of pharmacologic activity of guinea pig tracheal spirals and isolated parenchymal strips in vitro. Fedn Proc. Fedn Am. Sues exp. Biol. 36:494 (abs 1177). DuBols, A. B., BOTELnO,S. Y. and COMROE,J. H. JR. (1956a) A new method for measuring airway resistance in man using a body plethysmograph: values in normal subjects and in patients with respiratory disease. J. din Invest. 35: 327-335. DuBolS, A. B., BRODY,A. W., LEWIS, D. H. and BURGESS,B. F. JR. (1956b) Oscillation mechanics of lungs and chest in man. J. appl. Physiol. 8: 587-594. DUCKLES, S. P., RAYNER, M. D. and NADEL, J. A. (1974) Effects of CO 2 and pH on drug-induced contractions of airway smooth muscle. J. Pharmac. exp. Ther. 190: 472-481. ELLIOTT, E. A. and DAWSON, S. V. (1977) Test of wave-speed theory of flow limitation in elastic tubes. J. appl. Physiol. Resp. Environ. Exercise Physiol. 43: 516-522. ELLm, M. P. and LIVINGSTON,A. E. (1935) A method of directly recording changes in the calibre of the bronchi. J. Physiol. 89: 223-231. EMPEY, D. W., LAmNEN, L. A., JACOBS, L., GOLD, W. M. and NADEL, J. A. (1976) Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am. Rev. resp. Dis. 113: 131-139. ENGEL, L. A., LANDAU, L., TAUSSIG,L., MARTIN, R. R. and SYBRECHT,G. (1976) Influence ofbronchomotor tone on regional ventilation distribution at residual volume. J. appl. Physiol. 40:411-416. UNHORSING, G. (1977) Photography of peripheral pulmonary airway expansion as affected by surfactant. J. Appl. Physiol. Respirat. Environ. Exercise Physiol. 42: 976-979.

Methods of study of airway smooth muscle and its physiology

289

EYRE, P. (1969) The pharmacology of sheep tracheobronchial muscle: a relaxant effect of histamine on the isolated bronchi. Br. J. Pharmac. 36: 409-417. FARm, U, OTIs, A. B. and PROCTOR,D. F. (1957) Measurement ofintrapleural pressure at different points in the chest of the dog. J. Appl. Physiol. 10: 15-18. FARIDY, E. E. and PERMUTT,S. (1971) Surface forces and airway obstruction. J. Appl. Physiol. 30: 319-321. FARMER,J. B. and COLEMAN,R. A. (1970) A new preparation of the isolated intact trachea of the guinea pig. J. Pharm. Pharmac. 22: 46-50. FERREIRA,S. S. and VANE,J. R. (1967) Prostaglandins: their disappearance from and release into the circulation. Nature, Lond. 216: 868-873. FERRIS,B. G., MEAD,J. and Oem, L. H. (1964) Partitioning of respiratory flow resistance in man. J. appl. Physiol. 19: 653-658. FILLENZ, M. (1970) Innervation of pulmonary and bronchial blood vessels of the dog. J. Anat. 106: 449-461. F1NKLEMAN,B. (1930) On the nature of inhibition in the intestine. J. Physiol. 70: 145-157. FINUCANE,K. E., DAWSON,S. V., PHELAN,P. D. and MEAD,J. (1975) Resistance ofintrathoracic airways of healthy subjects during periodic flow. J. appl. Physiol. 38: 517-530. FISCHER, H. W. and BLAUG,S. M. (1969) Aerosol bronchography. Radiology 92:150-154. FISHER, A. B., DuBols, A. B. and HYDE, R. W. (1968) Evaluation of the forced oscillation technique for the determination of resistance to breathing. 3. din. Invest. 47: 2045-2057. FISHER, H. K., HOLTON, P., BUXTON,R. ST. J. and NADEL,J. A. (1970) Resistance to breathing during exerciseinduced asthma attacks. Am. Rev. resp. Dis. 101: 885-896. FISHER,H. K. and HANSEN,T, A. (1976) Site of action of inhaled 6 per cent carbon dioxide in the lungs of asthmatic subjects before and after exercise. Am. Rev. resp. Dis. 114: 861-870. FLEISCH,J. H., MALING,H. M. and BRODIE,B. B. (1970) Evidence for existence ofalpha-adrenergic receptors in the mammalian trachea. Am. J. Physiol. 218: 596-599. FOSTER, R. W. (1960) The paired tracheal chain preparation. J. Pharm. Pharmac. 12:189-191. FOSTER,R. W. (1966) The nature of the adrenergic receptors of the trachea of the guinea pig. J. Pharm. Pharmac. 18: 1-12. FOWLER, W. S. (1948) Lung function studies. II. The respiratory dead space. Am. J. Physiol. 154: 405-416. FRANK,N. R., AMDUR,M. O., WORCESTER,J. and WH1TTENBERGER,J. L. (1962) Effects of acute controlled exposure to SO 2 on respiratory mechanics in healthy male adults. J. appL Physiol. 17: 252-258. FRANK, N. R., MEAD, J. and FERRIS,B. G. JR. (1957) The mechanical behaviour of the lungs in healthy elderly persons. J. clin. Invest. 36: 1680-1687. FRANK, N. R., MEAD,J. and WHITTENBERGER,J. L. (1971) Comparative sensitivity of four methods for measuring changes in respiratory flow resistance in man. J. appl. Physiol. 31: 934-938. FRANZ,D. N. and IG~O, A. (1968) Conduction failure in myelinated and nonmyelinated axons at low temperatures. J. Physiol. 199: 319-345. FRANZ, D. N. and PERRY, R. S. (1974) Mechanisms for differential block among single myelinated and nonmyelinated axons by procaine. J. Physiol. 236:1193-210. FREEDMAN,B. J. (1972) The functional geometry of the bronchi. Bull. Physio-path. resp. 8: 545-551. FREEDMAN,S., TATTERSFIELD,A. E. and PruDE, N. B. (1975) Changes in lung mechanics during asthma induced by exercise. J. appl. Physiol. 38: 974-982. FROEB,H. F. and MEAD,J. (1968) Relative hysteresis of the dead space and lung in vivo. J. appl. Physiol. 25: 244-248. FRY, D. L. and HYATT,R. E. (1960) Pulmonary mechanics. A unified analysis of the relationship between pressure, volume and gasflow in the lungs of normal and diseased human subjects. Am. J. Med. 29: 672-689. FRY, D. L., STEAD,W. W., EBERT,R. V., LUBIN,R. I. and WELLS,H. S. (1952) The measurement ofintraesophageal pressure and its relationship to intrathoracic pressure. J. Lab. din. Med. 40: 664-673. GADDUM,J. H. (1953) The technique of superfusion. Br. J. Pharmac. 8: 321-326. GADDUM,J. H., HEBB,C. O., SILVER,A. and SWAN,A. A. B. (1953) 5-Hydroxytryptamine. Pharmacological action and destruction in perfused lungs. Quart. J. exp. Physiol. 38: 255-262. GADDUM,J. H. and STEPHENSON,R. P. (1958) A microbath. Br. J. Pharmae. 13: 493-497. GANDEVlA,B. (1963)The spirogram ofgross expiratory tracheobronchial collapse in emphysema. Quart. J. Med. 32: 23-31. GARDINER,A. J., WOOD,L., GAYRARD,P., MENKES,H. and MACKLEM,P. (1974) Influence of constriction in central or peripheral airways on maximal expiratory flow rates in dogs. J. appl. Physiol. 36: 554-560. GAYRARD,P., OREHEK,J., GRIMAUD,C. and CHARPIN,J. (1975) Bronchoconstrictor effects of a deep inspiration in patients with asthma. Am. Rev. resp. Dis. l l l : 433-439. GERBERSHAGEN,H. U. and BERGMAN,N. A. (1967) The effect of d-tubocurarine on respiratory resistance in anesthetized man. Anesthesiology 28: 981-984. GILES, R. E., FINKEL,M. P. and MAZUROWSKI,J. (1971) Use of an analog on-line computer for the evaluation of pulmonary resistance and dynamic compliance in the anesthetized dog. Arch. int. Pharmacodyn. Ther. 194: 213-222. GILLESPIE, D. J. and HYATT, R. E. (1974) Respiratory mechanics in the unanesthetized dog. J. appl. Physiol. 36: 98-102. GILLESPIE, D. J., LA1, Y. L. and HYATT, R. E. (1973) Comparison of esophageal and pleural pressures in the anesthetized dog. J. appl. Physiol. 33: 709-713. GOERG, R., ROECK,W. W. and MILNE, E. N. C. (1973) Tantalum oxide--a non-explosive substitute for metallic tantalum powder, lnvestig. Radiol. $: 333-338. GOLD, W. M., KESSLER,G.-F. and Yu, D. Y. C. (1972) Role of vagus nerves in experimental asthma in allergic dogs. J. appl. PhysioL 33: 719-725. GOLDEN, J. A., NADEL, J. A. and BousHEY, H. A. (1978) Bronchial hyperirritability in healthy subjects after exposure to ozone. Am. Rev. resp. Dis. 118: 287-294. GOLDMAN,M., KNUDSON,R. J., MEAD,J., PETERSON,N., SCHWABER,J. R. and WOVIL,M. E. (1970) A simplified measurement of respiratory resistance by forced oscillation. J. appl. Physiol. 28: ! 13-116.

290

H.L. HAHN and J. A. NADEL

GOLDMAN, E. and Puv, R. J. M. (1975) Effects of atropine on natural deflation flows and P-V curves of the isolated lung lobe. J. appl. Physiol. 38: 46-51. GRAF, P. D., FISCHER,S. P., NISAM,M., NADEL, J. A. and GOLD, W. M. (1975) An isolated tracheal segment to study airway reflexes in conscious dogs. Physiologist 18:23 I. GREEN, M. and MEAD, J. (1974) Time dependence of flow-volume curves. J. appl. Physiol. 37: 793-797. GREEN, M. and WIDDICOMBE,J. G. (1966) The effects of ventilation of dogs with different gas mixtures on airway calibre and lung mechanics. J. Physiol. 186: 363-381. GRIMBY, G., TAKISHIMA, T., GRAHAm, W., MACKLEM, P. and MEAD, J. (1968) Frequency dependence of flow resistance in patients with obstructive lung disease. J. clin. Invest. 47: 1455-1465. GRYGLEWSKI, R. J., DEMBINSKA-KIEC,A., GRODZINSKA, L. and PANCZENKO, B. (1976) Differential generation of substances with prostaglandin-like and thromboxane-like activities by guinea pig trachea and lung strips. In: Lung Cells in Disease (ed.) A. BouHoYS, pp. 289-307. Elsevier-North Holland, Amsterdam. GUIRGIS, H. M. (1969) The effect of adrenergic drugs on the isolated tracheal tube in vitro and on the bronchial resistance to inflation in vivo. Arch. Int. Pharmacodyn. Th&. 182: 147-160. HAHN, H, L., GRAF, P. D. and NADEL, J. A. (1976) Effect of vagal tone on airway diameters and on lung volume in anesthetized dogs. J. appl. Physiol. 41: 581-589. HAHN, H. L., WILSON, A. G., GRAF, P. D., FISCHER,S. P. and NADEL, J. A. (1978) Interaction between serotonin and efferent vagus nerves in dog lungs. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44: 144-149. HAKANSSON, C. H. and TOREMALM, N. G. (1967) Studies on the physiology of the trachea. IV. Electrical and mechanical activity of the smooth muscles. Ann. Otol. Rhinol. Laryngol. 76: 873-884. HANK1NSON, J. L. and LAPP, N. L. (1970) Time-pulse generator for flow-volume curves. J. appl. Physiol. 29: 109-110. HErin, C. (1969) Motor innervation of the pulmonary blood vessels of mammals. In: The Pulmonary Circulation and Interstitial Space, (eds.) A. P. FISHMANand H. H, HECHT, pp. 195-222. The University of Chicago, Chicago. HERZOG, H., KELLER, R., MAURER, W., BAUMANN, H. R. and NADJAFI, A. (1968) Distribution of bronchial resistance in obstructive pulmonary diseases and in dogs with artificially induced tracheal collapse. Respiration 25: 361-394. HICKEY, R. V., GRAF, P. D., NADEL, J. A. and LARSON, C. P. (1969)The effects of halothane and cyclopropane on total pulmonary resistance in the dog. Anesthesiology 31: 334-343. HILDEBRANDT,J. (1971) Lung hysteresis component of total pulmonary resistance in oscillatory methods. The Physiologist 14:162. H1MORI, N. and TAIRA, N. (1976) A method for recording smooth muscle and vascular responses of the bloodperfused dog trachea in situ. Br. J. Pharmac. 56: 293-299. HOLDEN, W. S. and ARDRAN,G. M. (1957) Observations on the movements of the trachea and main bronchi in man. J. Fac. Radiol. 8: 267-275. HOOKER, C. S., CALKINS, P. J. and FLEISCH, J. H. (1977) On the measurement of vascular and respiratory smooth muscle responses in vitro. Blood Vessels 14:1-11. HOPPIN, F. G., GREEN, I. D. and MEAD, J. (1969) Distribution of pleural surface pressure in dogs. J. appl. Physiol. 27: 863-873. HOPPlN, F. G. JR., GREEN, M. and MORGAN, M. S. (1978) Relationship of central and peripheral airway resistance to lung volume in dogs. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44: 728-737. HUGHES,J. M. B., HOPPIN, F. G. JR. and WILSON, A. G. (1972) Use of stereoscopic X-ray pairs for measurements of airway length and diameter in situ. Br. J. Radiology 45:477-4852 HUGHES, J. M. B., JONES, H. A. and WILSON, A. G. (1975) Bronchial hysteresis in excised lungs. J. Physiol. 249: 435-443. HUGHES,J. M. B., JONES,H. A., WILSON, A. G., GRANT, B. J. B. and PRIDE,N. B. (1974) Stability ofintrapulmonary bronchial dimensions during expiratory flow in excised lungs. J. appl. Physiol. 37: 684-694. HUTCHEON, M., GRIFFIN, P., LEVlSON,H. and ZAMEL,N. (1974) A new test in detection of mild abnormalities of lung mechanics. Am. Rev. resp. Dis. 110: 458-465. HUTCHEON, M. A., RODARTE, J. R. and HYATT, R. E. (1977) Effect of breathing helium-oxygen on static lung volumes and lung recoil in normal man. J. appl. Physiol. Resp. Environ. Exercise Physiol. 42: 899-902. HUTT, G. and WICK,H. (1956) Bronchiallumen und Atemwiderstand. Zeitschr. Aerosol Forsch. Ther. 5:131-140. HYATT, R. E. and FLATH, R. E. (1966) Influence of lung parenchyma on pressure-diameter behaviour of dog bronchi. J. appl. Physiol. 21: 1448-1452. HYATT, R. E. and WILCOX, R. E. (1961) Extrathoracic airway resistance in man. J. appl. Physiol. 16: 326-330. HYATT, R. E., ZIMMERMAN,I. R., PETERS,G. M. and SULLIVAN,W. J. (1970) Direct writeout of total respiratory resistance. J. appl. Physiol. 28: 675-678. INGRAM,R. H. JR. (1975) Effects of airway vs arterial CO2 changes on lung mechanics in dogs. J. appl. Physiol. 38: 603-607. IRAVANI,J. and SCHi3LER,K. G. (1971) In vitro Untersuchungen der Bronchomotorik der Ratte. Pneumonoloeie 144 : 253-607. JACKSON, A. C., BUTLER, J. D., MILLET, E. J., HOPPIN, F. G. JR. and DAWSON, S. V. (1977) Airway geometry by analysis of acoustic pulse response measurements. J. appl. Physiol. Resp. Environ. Exercise Physiol. 43: 523-536. JACKSON, A. C., MILLHORN, H. T. JR. and NORMAN, J. R. (1974) A reevaluation of the interrupter technique for airway resistance measurement. J. appl. Physiol. 36: 264-268. JAEGER, M. J. and OTIS, A. B. (1964) Measurement of airway resistance with a volume displacement plethysmograph. J. appl. Physiol. 19: 813-820. JAMES,G. W. L. (1969) The use of the in vivo trachea preparation of the guinea pig to assess drug action on lung. J. Pharm. Pharmac. 21: 379-386. JAMIE~SON,n . (1962) A method for the quantitative estimation of drugs on the isolated intact trachea. Br. J. Pharmac. 19: 286-294. JOHANSON,W. G. JR. and PIERCE, A. K. (1971) A noninvasive technique for measurement of airway conductance in

Methods of study of airway smooth muscle and its physiology

291

small animals. J. appl. Physiol. 30: 146-150. JONES, J. G., FRASER, R. B. and NADEL, J. A. (1975a) Prediction of maximum expiratory flow rate from areatransmural pressure curve of compressed airway. J. appl. Physiol. 38: 1002-1011. JONES, J. G., FRASER, R. B. and NADEL, J. A. (1975b) Effect of changing airway mechanics on maximum expiratory flow. J. appl. Physiol. 38: 1012-1021. JONSON, B. (1969) A method for determination of pulmonary elastic recoil and resistance at a regulated flow rate. Scand. J. clin. Lab. Invest. 24:115-125. JONSON, B. and BOUHUYS,A. (1967) Measurement of alveolar pressure. J. appL Physiol. 22: 1081-1085. JORDANOGLOU, J., KONTOS, J., PATELIS, N. and GARDIKAS, C. (1976) Mean nonelastic lung resistance and its equivalents (effective resistance). Pneumonologie 153: 209-215. JUNOD, A. F. (1977) Metabolism of vasoactive agents in lung. Am. Rev. resp. Dis. 115: 51-57. KAHN, R. H. (1907) Zur Physiologic der Trachea. Archivf~r Anatomie und Physiologie 398 426. KAPANCI, Y., ASSIMACOPOULOS,A., IRLE, C., ZWAHLEN,A. and GABB1ANI,O. (1974) Contractile interstitial cells in pulmonary alveolar septa: A possible regulator of ventilation-perfusion ratio. J. cell. Biol. 60: 375-392. KARCZEWSKI, W, and WIDDICOMBE, J. G. (1969) The effect of vagotomy, vagal cooling and efferent vagal stimulation on breathing and lung mechanics in rabbits. J. Physiol., Lond. 201: 259-270. KILBURN,K. H. (1960) Dimensional responses of bronchi in apneic dogs to airway pressure, gases and drugs. J. appl. Physiol. 15: 229-234. KIRKPATRICK, C. T. (1975) Excitation and contraction in bovine tracheal smooth muscle. J. Physiol. 244:263-281. KNUDSON, R. J. and KNUDSON, D. E. (1973) Pressure-flow relationships in the isolated canine trachea. J. appl. Physiol. 35:804 812. KNUDSON, R. J. and KNUDSON, D. E. (1975) Effect of muscle constriction on flow limiting collapse of isolated canine trachea. J. appl. Physiol. 38: 125-131. KOBLET, H. and WYSS, F. (1956) Das klinische und funktioneUe Bild des genuinen Bronchialkollapses mit Lungenemphysem. Helvetica Med. Acta 23: 553-560. KONZETT, H. and R~SSLER, R. (1940) Versuchsanordnung zu Untersuchungen an der Bronchialmuskulatur. Naunyn-Schmiedeberys Arch. Pharmakol. 195: 71-74. Koo, K. W., LEITH, D. E., SHERTER,C. B. and SNIDER, G. L. (1976) Respiratory mechanics in normal hamsters. J. appl. Physiol. 40: 936-942. KURES, H. (1974) An additive method for airway resistance measurement. Acta Paediat. scand. 63: 351-356. KURIYAMA, H. and ITO, Y. (1975) Recording ofintracellular electrical activity with microelectrodes. In: Methods in Pharmacoloyy, Volume 3, Smooth Muscle, pp. 201-230, (eds.) E. E. DANIELand D. M. PATON. Plenum Press, New York and London. LAITINEN, L. A., ELKIN, R. B., EMPEY,D. W., JACOnS,L., MILLS,J., GOLD, W. M. and NADEL, J. A. (1976a) Changes in bronchial reactivity after administration of live attenuated influenza virus. Am. Rev. resp. Dis. 113:194. LAITINEN, L. A., EMPEY, D. W., POPPIUS, H., LEMEN, R. J., GOLD, W. M. and NADEL, J. A. (1976b) Effects of intravenous histamine on static lung compliance and airway resistance in normal man. Am. Rev. resp. Dis. 114: 291-295. LANDAU, L. I. and PHELAN, P. D. (1973) Evaluation of two techniques for measurement of respiratory resistance by forced oscillation. A study in young subjects with obstructive lung disease. Thorax 28: 136-141. LAVELLE,T. F. JR., ROTMAN, H. H. and WEG, J. G. (1978) Isoflow-volume curves in the diagnosis of upper airway obstruction. Am. Rev. resp. Dis. 117: 845-852. LAVER, M. B., MORGAN,J., BENDIXEN,H. H. and RADFORD, E. P. JR. (1964) Lung volume compliance, and arterial oxygen tensions during controlled ventilation. J. appl. Physiol. 19: 725-733. LEDSOME,J. R., LINDEN, R. J. and NORMAN,J. (1971) The effect of light chloralose and pentobarbitone anaesthesia on the acid-base state and oxygenation of arterial blood in dogs. J. Physiol. 212:611-627. LEE, L. Y., BLEECKER,E. R. and NADEL,J. A. (1977) Effect of ozone on bronchomotor response to inhaled histamine aerosol in dogs. J. appl. Physiol. Resp. Environ. Exercise Physiol. 43: 626-631. LEMEN, R., BENSON, M. and JoNEs, J. G. (1974) Absolute pressure measurements with hand-dipped and manufactured esophageal balloons. J. appl. Physiol. 37: 600-603. LEOPOLD, J. G. and GOUGH, J. (1963) Post-mortem bronchography in the study of bronchitis and emphysema. Thorax 18: 172-177. LEUALLEN, E. C. and FOWLER, W. S. (1955) Maximal midexpiratory flow. Am. Rev. Tuberc. 72: 783-800. LLOYD, T. C. JR. (1963) Bronchoconstriction in man following single deep inspirations. J. appl. Physiol. 28: 114-116. LLOYD, T. C. JR. (1968) Hypoxic pulmonary vasonstriction: role of perivascular tissue. J. appl. Physiol. 25: 560-565. LLOYD, T. C. JR. and WRIGHT, G. W. (1963) Evaluation of methods used in detecting changes of airway resistance in man. Am. Rev. resp. Dis. 87: 529-537. LO, P. Y., ADAIKAN,P. G. and KARIM, S. M. M. (1976) Effects of PGA and PGB compounds on human respiratory tract smooth muscle in vitro. Prostaglandins II : 531-536. LOOFBOURROW,G. N., WOOD, W. B. and BAIRD, I. L. (1957) Tracheal constriction in the dog. Am. J. Physiol. 191 : 411-415. LULICH, K. M., MITCHELL, H. W. and SPARROW, M. P. (1976) The cat lung strip as an in vitro preparation of peripheral airways: a comparison of fl-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea. Br. J. Pharmac. 58: 71-79. LYNN JAMES,G. W. (1969) The use of the in vivo trachea preparation of the guinea pig to assess drug action on lung. J. Pharm. Pharmac. 21: 379-386. McDOUGAL, M. D. and WEST, G. B. (1953) The action of drugs on isolated mammalian bronchial muscle. Br. J. Pharmac. 8: 26-29. MCFADDEN, E. R. JR., INGRAM, R. H. JR., HAYNES, R. L. and WELLMAN, J. J. (1977) Predominant site of flow limitation and mechanisms of postexertional asthma. J. Appl. Physiol. Respirat. Environ, Exercise Physiol. 42; 746-752.

292

H. L. HAHNand J. A. NADEL

MACHT,D. I. and TING,G&H. (1921) A study of antispasmodic drugs on the bronchus. J. Pharmac. exp. Ther. 18: 373-398.

MCILROY,M. B., TIERNEY,D. F. and NADEL,J. A. (1963) A new method for measurement of compliance and resistance of lungs and thorax. J. appl. Physiol. 18:424-427. MACKLEM,P. T. and ENGEL,L. A. (1975) The physiological implications of airway smooth muscle constriction. Postgrad. Med. J. 51: Suppl. 7, 45-52. MACKLEM,P. T. and MEAD, J. (1967) Resistance of central and peripheral airways measured by a retrograde catheter. J. appl. Physiol. 22: 395-401. MACKLEM,P. T. and MEAD,J. (1968) Factors determining maximum expiratory flow in dogs. J. appl. Physiol. 25: 159-169.

MACKLEM,P. T. and WILSON,N. J. (1965) Measurement of intrabronchial pressure in man. J. appl. Physiol. 20: 653-663. MACKLEM,P.

T., WOOLCOCK, A. J., HOGG,J. C., NADEL,J. A. and WILSON,N. J. (1969) Partitioning of pulmonary resistance in the dog. J. appl. Physiol. 26: 798-805. MACKLIN,C. C. (1925) X-ray studies on bronchial movements. Am. J. Anaf. 35: 303-329. MCMAHON,S. M., PROCTOR, D. F. and PERMUTT,S. (1969) Pleural surface pressure in dogs. J. appl. Physiol. 27: 881-885.

MAIN,I. H. M. (1964) The inhibitory actions of prostaglandins on respiratory smooth muscle. Br. J. Pharmac. 22: 511-519.

MANN,S. P. (1971) The innervation of mammalian bronchial smooth muscle: the localization of catecholamines and cholinesterases. Histochemical J. 3: 319-331. MANSELL,A., DUBRAWSKY, C., LEVISON, H., BRYAN,A. C., LANGER,H., COLLINS-WILLIAMS, C. and ORANGE,R. P. (1974) Lung mechanics in antigen-induced asthma. J. appl. Physiol. 37: 297-301. MANSELL,A., LEVISON,H., KRUGER,K. and TRIPP,T. L. (1972) Measurement of respiratory resistance in children by forced oscillations. Am. Rev. resp. Dis. 106:710-714. MARSHALL,R. and HOLDEN,W. S. (1963) Changes in calibre of the smaller airways in man. Thorax 18: 5458. MATHI?, A. A. and HEDQVIST, P. (1975) Effect of prostaglandins F2. and E2 on airway conductance in healthy subjects and asthmatic patients. Am. Rev. resp. Dis. 111: 313-320. MEAD,J. (1960a) Control of respiratory frequency. J. appl. Physiol. 15: 325-336. MEAD,J. (1960b) Volume displacement body plethysmograph for respiratory measurements in human subjects. J. appl. Physiol. 15: 736-740.

MEAD,J. and COLLIER,C. (1959) Relation of volume history of lungs to respiratory mechanics in anaesthetized dogs. J. appl. Physiol. 14:669-678. MEAD,J., TURNER,J. M., MACKLEM,P. T. and LITTLE,J. B. (1967) Significance of the relationship between lung recoil and maximum expiratory flow. J. appl. Physiol. 22: 95-108. MEAD,J. and WHITTENBERGER, J. L. (1953) Physical properties of human lungs measured during spontaneous respiration. J. appl. Physiol. 5: 779-796. MEAD,J. and WHITYENBERGER, J. L. (1954) Evaluation of airway interrupter technique as a method for measuring pulmonary air-flow resistance. J. appl. Physiol. 6: 408-416. MELVILLE,K. I. and CAPLAN,H. (1948) Influence of lung distension upon the response of the bronchioles to epinephrine and to histamine. J. Pharmac. exp. Ther. 94: 182-191. MILIC-EWILI,J., MEAD,J., TURNER,J. M. and GLAUSER,E. M. (1964) Improved technique for estimating pleural pressure from esophageal balloons. J. appl. Physiol. 19: 207-211. MILLAR,R. A. and MORRIS,M. E. (1961) Sympatho-adrenal responses during general anaesthesia in the dog and man. Can. Anaes. Sot. J. 8: 356-386. MINK, S. and WOOD,L. D. H. (1978) How does HeO, cause increased maximum expiratory flow in excised human lungs? Am. Rev. resp. Dis. 117: 376. MURTAGH,P. S., PROCTOR,D. F., PERMUTT, S., KELLY,B. and EVERING,S. (1971) Bronchial closure withmecholyl in excised dog lobes. J. appl. Physiol. 31: 409-415. MUYSERS,K., SYIDT, U. and BUCHHEIM,F. W. (1969) Verbesserung ganzkijrperplethysmographischer Untersuchungen durch Einsatz eines Analogrechners. Pfiigers Arch. 307: 211-219. NADEL,J. A. (1970) New technique for studying structural changes of airways in vivo using powdered tantalum. In: Airway Dynamics, pp. 73-83, (ed.) A. Bouhuys. Thomas, Springfield, Illinois. NADEL, J. A., CABEZAS,G. A. and AUSTIN, J. H. M. (1971) In vivo roentgenographic examination of parasympathetic innervation of small airways. Invesfig. Radiol. 6: 9-17. NADEL,J. A., COLEBATCH, H. J. H. and OLSEN,C. R. (1964) Location and mechanism of airway constriction after barium sulfate microembolism. J. appl. Physiol. 19: 387-394. NADEL,J. A., SALEM,H., TAMPLIN,B. and TOKIWA,Y. (1965) Mechanism ofbronchoconstriction during inhalation of sulfur dioxide. J. appl. Physiol. 20: 164167. NADEL,J. A. and TIERNEY,D. F. (1961) Effect of a previous deep inspiration on airway resistance in man. J. appl. Physiol. 16: 717-719. NADEL,J. A. and WIDDICOMBE, J. G. (1962a) Reflex effects of upper airway irritation on total lung resistance and blood pressure. J. appl. Physiol. 17: 861-865. NADEL,J. A. and WIDDICOMBE, J. G. (1962b) Effect of changes in blood gas tensions and cartoid sinus pressure on tracheal volume and total lung resistance to airflow. J. Physiol. 163: 13-33. NADEL,J. A., WOLFE,W. G. and GRAF,P. D. (1968) Powdered tantalum as a medium for bronchography in canine and human lungs. Investig. Radiol. 3: 229-238. NEERGAARD VON K. and WIRZ, K. (1927a) Uber eine Methode zur Messung der Lungenelastizitlt am lebenden Menschen, insbesondere beim Emphysem. Z. Klin. Med. 105: 35-50. NEERGAARD VON,K. and WIRZ, K. (1927b) Die Messung der Stromungswiderstiinde in den Atemwegen des Menschen, insbesondere bei Asthma und Emphysem. Z. Klin. Med. 105:51-82. NEWHOUSE, M. T., BECKLAKE, M. R., MACKLEM,P. T. and MCGREGOR,M. (1964) Effect of alterations in end-tidal CO, tension on flow resistance. J. appl. Physiol. 19: 745-749.

Methods of study of airway smooth muscle and its physiology

293

NISSELL, O. (1950) The action of oxygen and carbon dioxide on the bronchioles and vessels of the isolated perfused lungs. Acta Physiol. scand. 21: Suppl. 73, 5-62. NIgSELL, O. I. and DtJBols, A. B. (1954) Relationship between compliance and FRC of the lungs in cats, and measurement of resistance to breathing. Am. J. Physiol. 178:206-210. NISELL, O. I. and ErmNER, U S. G. (1956) A simple apparatus for measurement of pressure volume relationship in respiration. J. appl. Physiol. 8: 565-567. NISELL, O. and EHRNER, L. (1958) The resistance to breathing determined from time-marked respiratory pressure volume loops. Acta Med. Scand. 161: 427-436. NOLTE, D. (1969) Experimental studies on the existence of isothermal conditions in the human lung. Int. Symposium on body plethysmography, Nijmegen. Progr. Resp. Res. 4: 102-108. OFFERMEIER,J. and ARIENS,E. J. (1966) Serotonin. 1. Receptors involved in its action. Arch. int. Pharmacodyn. Ther. 164: 192-215. OLIVE, J. T. JR. and HYATT, R. E. (1972) Maximal expiratory flow and total respiratory resistance during induced bronchoconstriction in asthmatic subjects. Am. Rev. resp. Dis. 106: 366-376. OLSEN, C. R., COLEBATCH, H. J. H., MEnEL, P. E., NADi/L, J. A. and STAUB, N. C. (1965) Motor control of pulmonary airways studied by nerve stimulation. J. appl. Physiol. 20: 202-208. OLSEN, C. R., DEKOCK, M. A. and COLEBATCH,H. J. H. (1967a) Stability of airways during reflex bronchoconstriction. J. appl. Physiol. 23: 23-26. OLSEN, C. R., STEVENS, A. E. and MCILROY, M. B. (1976b) Rigidity of trachea and bronchi during muscular constriction. J. appl. Physiol. 23: 27-34. OLSEN, C. R., STEVENS,A. E., PRIDE, N. B. and STAUB,N. C. (1967c) Structural basis for decreased compressibility of constricted tracheae and bronchi, d. appl. Physiol. 23: 35-39. OREHEK, J., DOUGLAS, J. S. and BOUHUYS,A. (1975a) Contractile responses of the guinea pig trachea in vitro : modification by prostaglandin synthesis--inhibiting drugs. J. pharmac, exp. Ther. 194: 554-564. OREHEK, J., GAYRARD, P., GRIMAtrD, C. and CHARPIN, J. (1975b) Effect of maximal respiratory manoeuvres on bronchial sensitivity of asthmatic patients as compared to normal people. Br. Med. J. 1:123-125. OREHEK, J., GAYRARD, P., SMITH,A. D., GRIMAUD, C. and CHARPIN, J. (1977) Airway response to carbachol in normal and asthmatic subjects. Am. Rev. resp. Dis. 115: 937-943. OTIS, A. B., MCKERROW, C. B., BARTLETT,R. A., MEAD,J., MCILROY, M. B., S~LVERSTONE,N. J. and RADFORD, E. P. (1956) Mechanical factors in distribution of pulmonary ventilation. J. appl. Physiol. 8: 427-443. PAINTAL, A. S. (1973) Vagal sensory receptors and their reflex effects. Physiological Reviews 53: 159--227. PALOMBINI, B. and CoBuglq, R. F. (1972) Control of the compressibility of the canine trachea. Resp. Physiol. 15: 365-383. PARE, R. D., BOUCHER, R., MICHOUD, M.-C. and HOGG, J. C. (1978) Static lung mechanics of intact and excised rhesus monkey lungs and lobes. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44: 547-552. PARKER, J. C., PETERS, R. M. and BARNETT,T. B. (1963) Carbon dioxide and the work ofbreathing. J. clin. Invest. 42: 1362-1372. PARRISIUS, G. S. W,, KAISER, W. P., LEUCHT, K. and MARSCHALL, M. (1972) Die Inhalationsbronchographie im Tierexperiment mit jodierten Kontrastmittelpulvern. Fortschr. R~ntgenstr. 117: 1-8. PATTERSON, R. (1958) The tracheal strip: observations on the response of tracheal muscle. J. Allergy 29: 165-172. PERSSON, C. G. A. and EKMAN, M. (1976) Contractile effects of histamine in large and small respiratory airways. Agents & Actions 6: 389-393. PLoY-SONG-SANG, Y., CORBIN, R. P. and ENGEL, L. A. (1978) Effects of intravenous histamine on lung mechanics in man after beta blockade. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44: 690-695. PRICE, H. L., LINDE, H. W., JONES, R. E., BLACK, G. W. and PRICE, M. L. (1959) Sympatho-adrenal responses to general anesthesia in man and their relation to hemodynamics. Anesthesiology 20: 563-575. PRIDE, N. B., GRAF, P. D. and NADEL, J. A. (1964) Site of maximal expiratory flow limitation in cats. The Physiologist 7: 228. PRIDE, N. B., PERMUTT, S., RILEY, R. L. and BROMBERGER-BARNEA,B. (1967) Determinants of maximal expiratory flow from the lungs. J. appl. Physiol. 23: 646-662. PUN, L.-Q., ATKiNSON, J. M. and RANO, M. J. (1971) Reversal of the bronchodilator action of ephedrine during ventilation with carbon dioxide. European J. Pharmac. 15:110-118. o RADFORD, M. (1974) Measurement of airway resistance and thoracic gas volume in infancy. Arch. Dis. Child. 49: 611-615. RICHARDSON,J. and Bi/LAND,J. (1976) Nonadrenergic inhibitory nervous system in human airways. J. appl. Physiol. 41: 764-771. RICHARDSON,J. B. and BOUCHARD,T. (1975) Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. J. Allergy 56: 473-480. RICHARDSON, J. A., WOODS, E. F. and RICHARDSON, A. K. (1957) Plasma concentrations of epinephrine and norepinephrine during anesthesia. J. Pharmac. exp. Ther. 119: 378-384. ROBINSON, N. E. and GILLESPIE, J. R. (1973) Lung volumes in aging beagle dogs. J. appl. Physiol. 35: 317-321. RODARTE, J. R. and HYATT, R. E. (1973) Effect of acute exposure to CO 7 on lung mechanics in normal man. Resp. Physiol. 17: 135-145. ROSA, L. M. and McDOWALL, J. S. (1951) The action of the local hormones on the isolated human bronchus. A cta alleryoloyica IV: 293-304. ROY, C. S. and BROWN, G. (1885) On bronchial contraction. J. Physiol. 6: 21-25. RLrBINFELD,A. R. and PAIN, M. C. F. (1977) Relationship between bronchial reactivity, airway caliber, and severity of asthma. Am. Rev. resp. Dis. 115: 381-387. RUFFIN, R. E., DOLOVlCH, M. B., WOLFF,R. K. and NEWHOUSE, M. T. (1978) The effects of preferential deposition of histamine in the human airway. Am. Rev. resp. Dis. 117: 485-492. SACKNER,M. A., FEISAL, K. A. and DuBoIs, A. B. (1964) A bag-in-box system for maintaining gases at body temperature within the body plethysmograph. J. appL Physiol. 19: 534-535. SAMPSON,S. R. and VIDRUK,E. H. (1975) Properties of'irritant' receptors in canine lungs. Respiration Physiol. 25: 9-22.

294

H.L. HAHN and J. A. NADEL

SASAKI,H., TAKISHIMA,T. and SASAKI,T. (1977) Influence of lung parenchyma on dynamic bronchial collapsibility of excised dog lungs. J. appl. Physiol. Resp. Environ. Exercise Physiol. 42:699 705. SAUNDERS,N. A., BEXTS,M. F., PENGELLY,L. D. and I~BUCK, A. S. (1977) Changes in lung mechanics induced by acute isocapnic hypoxia. J. appl. Physiol. Resp. Environ. Exercise Physiol. 42: 413-419. SCHILD, H. O., HAWKINS, D. F., MONGAR,J. L. and HERXHEIMER,H. (1951) Reactions of isolated human asthmatic lung and bronchial tissue to a specific antigen. Histamine release and muscular contraction. Lancet 261: 376-382. SCHILDER, D. P., ROBERTS,A. and FRY, D. L. (1963) Effect of gas density and viscosity on the maximal expiratory flow-volume relationship. J. din. Invest. 42:1705-1713. SCHWIELER, G. H. (1966) The effect of vagal stimulation on the bronchial tree in the cat. Acta. physiol, scand. 66: 495-500. SEVERINGHAUS,J. W. and STUPFEL, M. (1955) Respiratory dead space increase following atropine in man, and atropine, vagal or ganglionic blockade and hypotherrnia in dogs. J. appl. Physiol. 8: 81-87. SEVERINGHAUS, J. W., SWENSON, E. W., FINLEY, T. N., LATEGOLA, M. T. and WILLIAMS, J. (1961) Unilateral hypoventilation produced in dogs by occluding one pulmonary artery. J. appl. Physiol. 16: 53-60. SIMONSSON,B. G., JACOBS,F. M. and NADEL, J. A. (1967) Role of autonomic nervous system and the cough reflex in the increased responsiveness of airways in patients with obstructive airway disease. J. din. Invest. 46: 1812-1818. SLADEN,A., LAVER,M. B. and PONTOPPIDAN,H. (1968) Pulmonary complications and water retention in prolonged mechanical ventilation. N. Engl. J. Med. 279: 448-453. ~MEJKAL,V. and PALE(2EK,F. (1972) Determination of airway resistance in rats and guinea pigs. Physiol. Bohemoslov. 21: 563-568. SMIDT, U., FINKENZELLER, P. and RENNINGS, C. (1975) On-line Computereinsatz in der Ganzk6rperplethysmographie zur Berechnung der mittleren Resistance. Pneumonologie 151: 223-231. SMIDT, U., MUYSERS,K. and BucnI-miM, W. (1969) Electronic compensation of differences in temperature and water vapour between in- and expired air and other signal handling in body plethysmography. Int. Symposium on Body Plethysmography Nijmegen. Prog. Resp. Res. 4: 39-49. SMITh, J. C., SXITIK,F. P. and SwIFt, D. L. (1976) Airway visualization by tantalum inhalation bronchography. Am. Rev. resp. Dis. 113: 515-529. SOBOL, B. J. (! 970) A simple, rapid technique for assessing airway resistance during quiet breathing. Am. Rev. resp. Dis. 102: 970-974. SOLLMANN,T. and GILBERT,A. J. (1937) Microscopic observations ofbronchiolar reactions. J. Pharmac. exp. Ther. 61: 272-285. SOMLYO, A. P. and SOMLYO,A. V. (1970) Biophysics of smooth muscle excitation and contraction. In: Airway Dynamics, pp. 209-228, (ed.) A. BountJvs. Charles C. Thomas, Springfield, Illinois. SoNr,m, C. (1923) Untersuchungen fiber die relative Weite der Bronchiolen bei der verschiedenen Luftspannung der Lnngen. Acta Med. scand. 38:313 341. SOUHRADA,J. F. and DICKEY, D. W. (1976) Mechanical activities of trachea as measured in vitro and in vivo. Resp. Physiol. 26: 27-40. SPRIGGS,T. L. B. (1965) The effect of anaesthesia induced by urethane or phenobarbitone upon the distribution of peripheral catecholamines in the rat. Br. J. Pharmac. 24: 752-758. STANESCU, D. C., DE SUTTER, P. and VAN DE WOESTIJNE, K. P. (1972a) Pressure-corrected flow body plethysmograph. Am. Rev. resp. Dis. 105: 304-305. STAN~CU, D. C., FESLER, R., VERITER, C., FRANS, A. and BRASSEUR, L. (1975) A modified measurement of respiratory resistance by forced oscillation during normal breathing. J. appl. Physiol. 39:305-311. STA~SCU, D. C., PATTIJU, J., CLi~MENT, J. and VAN DE WOESTImE, K. P. (1972b) Glottis opening and airway resistance. J. appl. Physiol. 32: 460-466. STASZEWSKA-BARCZAK,J. and VANE, J. R. (1965) The release of catechol amines from the adrenal medulla by histamine. Br. J. Pharmac. 25: 728-742. STAtJB,N. C. and STOREY,W. F. (1962) Relation between morphological and physiological events in lung studied by rapid freezing. J. appl. Physiol. 17: 381-390. ST~PHENS,N. L. (1975) Physical properties of contractile systems. In: Methods in Pharmacology, Volume 3, Smooth Muscle, pp. 265-296, (eds.) E. E. DANIEL and D. M. PATON. Plenum Press, New York and London. STEPHENS,N. L., KROEGER, E. and MErtTA, J. A. (1969) Force-velocity characteristics of respiratory airway smooth muscle. J. appl. Physiol. 26: 685-692. STEPHENS,N. L., MEVERS,J. L, and CHERNlaCK, R. M. (1968) Oxygen, carbon dioxide, H + ion, and bronchial lengthtension relationships. J. appl. Physiol. 25: 376-383. STERLING, G. M. (1968) The mechanism ofbronchoconstriction due to hypocapnia in man. Clin. Sci. 34:277 285. STERLING, G. M. (1969) The mechanism of decreased specific airway conductance in man during hypercapnia caused by inhalation of 7 per cent CO 2. Clin. Sci. 37: 539-548. STERLING, G. M., HOLST, P. E. and NADEL, J. A. (1972) Effect of CO~ and pH on bronchoconstriction caused by serotonin vs acetylcholine. J. appl. Physiol. 32: 39-43. STOCKS, J., LEVY, N. M. and GODFREY, S. (1977) A new apparatus for the accurate measurement of airway resistance in infancy. J. appl. Physiol. Resp. Environ. Exercise Physiol. 43:155-159. STRANSKY, A., SZEREDA-PRZ~STASZEWSKA,M. and WIDDiCOMBE, J. G. (1973) The effects of lung reflexes on laryngeal resistance and motoneurone discharge. J. Physiol. 231: 417-438. STm~CKER, E.-P., WOLVART,W. and KNOnLOOt, S. (1974) Vergleicbende bronchographische Untersuchungen mit Tantalstaub und wfissrigem Kontrastmittel. Radiologe 14: 506-509. STUTZ, E. (1950) Bronchographische Beitr~ige zur normalen und pathologischen Physiologie der Lungen. Fortschr. R6ntgenstr. 72: 309-338. SWENSON, E. W., FINLEY, T. N. and GUZMAN, S. V. (1961) Unilateral hypoventilation in man during temporary occlusion of one pulmonary artery. J. clin. Invest. 40: 828-835. TOSEV, J., DALEY, L. and HILTON, A. (1969) A method for recording intrapleural pressure in conscious,

Methods of study of airway smooth muscle and its physiology

295

semirestrained mammals. J. appl. Physiol. 27: 902-903. T~NDELENBUR~, P. (1912) Physiologische und pharmakologische Untersuchungen an der isolierten Bronchialmuskulatur. Arch. Exp. Path. Pharmak. 69: 79-107. T/2RICER, R. K. and ZENCIL, H. (1976) Release of prostaglandin-like material from isolated cat tracheal muscle by electrical and mechanical stimulation. Arch. Int. PhysioL Biochim. 84: 833-841. UgiL, R. R. and LEWlS, F. J. (1974) Digital computer calculation of human pulmonary mechanics using a least squares fit technique. Computers Biomedical Res. 7: 489-495. VAN DE WOESTIJNE. K. P. and BouIJuYs, A. (1969) Spirometer response and pressure correction in body plethysmography. Int. Symposium on Body Plethysmography Nijmegen. Prog. Resp. Res. 4: 64-74. VIDRUK, E. H., HAHN, H. L., NADEL, J. A. and SAMPSON,S. R. (1977) Mechanisms by which histamine stimulates rapidly adapting receptors in dog lungs. J. appL Physiol. Resp. Environ. Exercise Physiol. 43: 397-402. VINCENT, N. J., KNUDSON, R., LEITH, D. E., MACKLEM,P. T. and MEAD, J. (1970) Factors influencing pulmonary resistance. J. appl. physiol. 29: 236-243. VUILLEUMIER,P. (1944) Uber eine Methode zur Messung des intraaveol/iren Druckes und der Strbmungswiderstiinde in den Atemwegen des Menschen. Z. klin. Med. 143: 598-717. WANNER, A. and SACKNER, M. A. (1973) Transvenous phrenic nerve stimulation in anesthetized dogs. J. appL Physiol. 34: 489-494. WASSERMAN, M. A. (1975) Bronchopulmonary responses to prostaglandin F2a, histamine and acetylcholine in the dog. Europ. J. Pharmac. 32: 146-155. WATSON, H., LANDA~J. and SACKNER,M. A. (1975) Automated system for measurement of mechanics of breathing. Med. Instrum. 9: 3-10. WELLMAN,J. J., BROWN, R., INGRAM,R. H. JR., MEAD,J. and MCFADDEN, E. R. JR. (1976) Effect of volume history on successive partial expiratory flow-volume manoeuvers. J. appL Physiol. 41: 153-158. WICK, H. (1952) l~ber die Wirkung der Kohlens~iure auf daN Trachealpriiparat des Hundes. Arch. Int. Pharmacodyn. Ther. 88: 450-457. WIDDICOMBE, J. G. (1977) Studies on afferent airway innervation. Am. Rev. resp. Dis. 115: 99-105. WIDDICOMBE,J. G. and NADEL,J. A. (1963a) Reflex effects of lung inflation on tracheal volume. J. appl. Physiol. 18: 681-686. WIDDICOMaE, J. G. and NADEL, J. A. (1963b) Airway volume, airway resistance, and work and force of breathing: theory. J. appl. Physiol. 18: 863-868. WOHL, M. E. B., TURNER,J. and MEAD,J. (1968) Static volume-pressure curves of dog lungs--in vivo and in vitro. J. appl. Physiol. 24: 348-354. WDHL, M. E. B., STIGOL, L. C. and MEAD, J. (1969) Resistance of the total respiratory system in healthy infants and infants with bronchiolitis. Pediatrics 43: 495-509. WOOD, L. D. H. and BRYAN,A. C. (1969) Effect of increased ambient pressure on flow-volume curve of the lung. J. appL Physiol. 27: 4-8. WOOD, L. D. H., ENGEL, L. A., GRIFFIN, P., DE.SPAS,P. and MACKLEM,P. T. (1976) Effect of gas physical properties and flow on lower pulmonary resistance. J. appL Physiol. 41: 234-244. WOOD, L. D. H. and ZEISMANN,M. M. (1976) Why does reduced gas density cause increased maximum expiratory flow? Fedn Proc. Fedn Am. Socs exp. Biol. 35: 231. WOOLCOCK, A. J., MACKLEM,P. T., HOGG, J. C., WILSON, N. J., NADEL, J. A., FRANK, N. R. and BRAIN,J. (1969a) Effect of vagal stimulation on central and peripheral airways in dogs. J. appl. Physiol. 26: 806-813. WOOLCOCK, A. J., MACKLEM,P. T., HOOG, J. C. and WmSON, N. J. (1969b) Influence of autonomic nervous system on airway resistance and elastic recoil, J. appl. Physiol. 26: 814-818. WRIGHT, B. M. and MCKARROW, C. B. (1959) Maximum forced expiratory flow rate as a measure of ventilatory capacity. Br. Med. J. 2: 1041-1047. YAMAGUCHI, T., HITZIG, B. and COaURN, R. F. (1976) Endogenous prostaglandins and mechanical tension in canine trachealis muscle. Am. J. Physiol. 230: 1737-1743. YEATES,D. B., ASPIN, N., LEVISON,H., JONES, M. T. and BRYAN,A. C. (1975) Mucocociliary tracheal transport rates in man. J. appl. Physiol. 39: 487-495. YOSmDA, S. (1964) Effects of bronchoactive agents on static pressure-volume characteristics of dog lungs. Am. J. Physiol. 206: 321-326. YOUNES, M. and YOUSSEF,M. (1978) Effect of five human anesthetics on respiratory control in cats. J. appl. Physiol. Resp. Environ. Exercise Physiol. 44: 596-606. YOUNG, A. C. (1955) Dead space at rest and during exercise. J. appl. PhysioL 8: 91-94.