Re.\p;rurion 0
t’/~r.sio/o~~~ ( 1977) 31, 365 -375
El.sevierNorth-Holland
Biomedical
Press
THE TRANSDUCTION PROPERTIES OF TRACHEAL, STRETCH KECEPTOKS fh’ I’ITKU
Abstract.
Extrathoracic
trachea
was stretched.
longitudinal
or transverse
as the tension decreased fusion showing
tracheal
stretch
The receptors
receptors
responded
axis of the trachea.
and tracheal
frequency
fluid. Contraction that the receptors
but the receptors
of tracheal studied
muscle.
Induced
Acetylcholine Dog
component
in the response.
were not affected
were effectively
Discharge
were recorded
frequency
by changes
by acetylcholine.
br r.i/ro when the
by displacement
They were sensitive to the rate of change
level Itself. i.c. there was a dynamic
dtscharge
tension
to an increase in tension produced
in the
of tension as well
A fall in temperature
in P,,,? and pH of the per-
increased
discharge
frequency
m series with the muscle. Temperature Tracheal
muscle
Tracheal
receptor
It is now thought that many of the ‘pulmonary’ stretch receptors first studied in detail by Adrian (1933) and extensively investigated since (see Widdicombe, 1974, for review). are in the trachea and main bronchi and not in the lung (Miserocchi (‘i a/.. 1973).The transduction properties of tracheal stretch receptors are therefore of interest. since they may well be similar to the properties of stretch receptors throughout the respiratory tree. The tracheal stretch receptors arc found in the fibromuscular band which holds the dorsat ends of the tracheal rings together (Bartlett cf trl., 1976a). Although present in greatest number at the lower end of the trachea, near the bifurcation, they arc also found in the extrathoracic trachea (Bartlett c’t trl., 1976a). Like stretch receptors throu~hoLit the respiratory tract they respond to a change in airway pressure as welt as to airway pressure itself(Bartlett et ul., t 976b). However, airway pressure is ;I very indirect measure of the stimulus to which the receptors respond.
ci.
366
W.
BRADLEY
They are likely to be more directly of the airways. This paper describes measured
in a tracheal
preparation
AND
N. SCHEURMIER
influenced by changes of tension in the walls an in vitro technique by which tension can be directly
whilst
stretch
receptor
discharge
is
recorded.
Methods
Sixteen dogs were anaesthetized with pentobarbitone (30 mg/kg i.v.) and the extrathoracic trachea separated from surrounding structures, taking care not to damage its nerve supply. A lethal dose of pentobarbitone was then given, and the trachea quickly removed, the lower transection being made as far down the trachea as possible from a neck approach (usually between 3 and 4 cm from the tracheal bifurcation). The tracheal rings were cut through on the ventral surface and the preparation immersed in Kreb’s solution with the following composition in mMo1.L~ ‘-Nat, 142.9; K+, 5.92; Ca++, 2.54; Mg++, 1.18; Cl’, 125.3; SO:, 1.18; H,PO;, 1.18; HCO;, 24.9; glucose 11.1. Figure I illustrates a cross-section of the perspex perfusion chamber and associated apparatus. Both strips of cartilage were gripped by stainless steel jaws. On one side the jaws were attached to a micromanipulator and on the other side to a Devices force transducer (type 4150). The transducer was attached to the pulling arm of a Ling puller (type 202) which was driven by a Crown Amplifier (DC-300 A) controlled by a waveform generator (Feedback, TWG 500). By this means it was possible to produce displacement in the transverse axis of the trachea, and measure the resulting tension. Tension changes in the longitudinal axis of the trachea were made in one experiment by applying the jaws across the preparation, i.e. at right angles to the usual method.
TAPE PAPER
STRAIN
RECORDER
8
RECORDER
CUAGE MICROMANIPUL
Fig.
I.
Cross-sectional
diagram of perfusion chamber and associated apparatus. tion.
ATOR
See text for full descrip-
TRACHEAL
Fluid
from the perfusion
STRETCH
chamber
RECEPTORS
was filtered
367
in l?itro
and pumped
to a reservoir
where
it was oxygenated with 7 II;, CO2 in oxygen. This kept the PcoJ and pH at physiological levels. In some experiments these were changed by altering the CO1 concentration in the equilibrating gas. The fluid then flowed by gravity through a heat exchanger back into the perfusion chamber. The temperature of the fluid was normally kept at 37 C by means of the heat exchanger and water jacket around the perfusion chamber. The fluid in the heating circuit was warmed and circulated by means of a Churchill pump. Liquid paraffin floating on top of the Kreb’s solution in the perfusion chamber was prevented from being circulated by means of a baffle plate. Nerves coming out of the fibromuscular band were brought up into the liquid paraffin for recording purposes and were split with watchmakers’ forceps until single unit activity was obtained. The action potentials were recorded from platinum electrodes and amplified with a Hewlett Packard amplifier (881 IA). The Devices transducer was energized from a Hewlett Packard module (SSOSB). Both signals were displayed on a Medelec recorder and also stored on tape (Tandberg, series 100). Impulse frequency was measured either by hand or from a frequency impulse meter. The PcoI, PO2 and pH of the perfusion fluid were measured using a Corning EEL blood gas analyzer (type 165). All single unit activity recorded was from fibres showing slow adaptation to stretch. More rapidly adapting units were seen on occasions, but were not studied in detail. Most experiments were terminated because of the lack of remaining unsplit nerve tibres, usually about nine hours after the removal of the trachea from the animal. The receptors remained active and stable over this period. The Ling puller has a maximum stroke of 5 mm and, therefore, changes in length were no greater than this and were usually smaller. Measurements of length change were not made during the experiment, but a length-tension curve was made after
the experiment. The relation between length and tension was curvilinear, the increase in tension for a given displacement rising sharply as the initial tension was increased. The initial tension was varied during the experiment by resetting the micromanipulator, but most studies were made with the highest tension not exceeding 200 g
applied to 9 cm of trachea.
Results Response to stretch
in tension and a rise in discharge frequency in all receptors studied. A square wave increase in tension produced a rapid increase in discharge frequency which then declined slowly although the tension was maintained, i.e. the receptor adapted. A sudden reduction in tension was usually associated with cessation of discharge, but activity returned even though the tension did not change. The receptors were thus sensitive to a change in tension Displacement
in the transverse
direction
produced
an increase
368
G. W. BRADLEY
AND
N. KHEUKMIEH
6Or
0
Fig. 2. Relation
L5
between discharge
frequency
,
50 TENSION
and tracheal
75 (g)
tension
for a single stretch
receptor
in steady-
state conditions.
as well as to the level of tension. An example of response to stretch
in virtually
steady
state conditions
is shown,
for a single stretch receptor, in fig. 2. Discharge frequency was measured over the period 4-5 seconds after a sudden increase in tension, when adaptation had taken
0.1 Hz
0.6
Hz
1 .O Hz
4.0
Hz
DISCHARGE FMOUE NCV IttZ) TENSION ‘d
6o 25
.,
Fig. 3. Response
of a single tracheal
stretch receptor
to sinusoidal
changes
:._ ‘.
in tension.
_.., ‘.
:., . .
The lower trace in
each record shows the action potentials, the middle trace shows the tension changes and the upper trace shows the discharge frequency as measured by a frequency impulse meter. The response at 4 different sinusoidal frequencies is shown, 0. I, 0.6, I .O and 4.0 Hz. The time scale is different in each case, and is given by the period
of the sine wave which is the reciprocal
of the sinusoidal
frequency.
TRACHEAL
place. The relationship linear and the minimum
STRETCH
between
tension
discharge
RECEPTORS
and discharge
frequencies
iti ritro
369
frequency
varied between
was usually
curvi-
IO and 30 Hz in the
majority of cases. Sinusoidal changes in tension were used to analyse the frequency response of the receptors (fig. 3). In such circumstances the peak frequency of discharge preceded the tension peak, and this phase lead became larger as the frequency of the sinusoid increased. The peak frequency of discharge often increased as the sinusoidal frequency increased, even though the changes in tension were kept constant. This is clearly seen in fig. 3 (note that the time scale is quite different for each record). Figure 4 shows the relationship between phase lead and sinusoidal frequency for 9 different receptors. The mean and standard error are shown for the same data in the lower graph. The phase lead increased at the higher sinusoidal frequencies, although this was not obvious until frequencies above 0.4 Hz were reached. One receptor appeared to show a sharp decline in phase lead with increase in sinusoidal frequency until a minimum was reached at 0.6 Hz. The response of this receptor was unique in that its discharge frequency profile was not sinusoidal at the lower frequencies of oscillations. The peak discharge frequency was reached early and then fell slowly until the tension started decreasing. Figure 5 shows the peak discharge frequencies at different sinusoidal frequencies for the same 9 receptors (with mean and standard error in the lower graph). The peak discharge frequency increased with increase in sinusoidal frequency, although the average
effect was quite small. 100
60
01 01
02
04
06
1.0
20
64
WAVE FREQUENCY (Hz) Fig. 4. Relation between phase lead and sine wave frequency for 9 different receptors. Top graph individual results and lower graph shows mean and one standard error for the same data. SINE
shows
370
G. W. BRADLEY
AND N. SCHEURMIER
I
81 Fig, 5. Relation fig. 5). Top graph
between
02
peak discharge
shows individual
o-1
06
1.0
20
SINE WAVE FREQUENCY (Hz) frequency and sine wave frequency
results and lower graph
5.0
for 9 receptors
shows mean and one standard
(same as in error for the
same data.
With the steel jaws applied in the usual way it was impossible to produce a satisfactory increase in tension by pulling in the longitudinal direction. When this was attempted by gripping both ends of the tibromuscular band with forceps and pulling, a fall in discharge frequency was obtained unless transverse shortening was prevented by firmly fixing the puller arm. In these circumstances no significant change in discharge frequency was seen, but the actual tension changes at the receptor site would be small because of the splinting effect of the steel jaws. When a longitudinal pull was produced locally, by means of a hook on both sides of the receptor site, the receptor proved sensitive to longitudinal displacement. This was confirmed by applying the steel jaws across the trachea. Longitudinal displacement could then be produced satisfactorily and discharge frequency increased with tension even though transverse shortening occurred. Adaptation to sudden changes in tension was also seen when displacement was produced in the longitudinal direction.
Ejjkt of’ CO, Changes in perfusion fluid Pee,, with concomitant changes in pH, had no effect on tracheal stretch receptor discharge. This is shown in fig. 6 for 16 receptors, the
TRACHEAL
STRETCH
RECEPTORS
PERFUSION
Fig. 6. Relation
between discharge
30
20
10
frequency
FLUID
and perfusion
LO
Pc02
371
itl l-ift-0
50
(mm Hg)
fluid Pto2 for I6 different
receptors.
See text
for details.
frequency of discharge being measured at a low and normal value of Pco, without changing the tension. In these circumstances changing the Pcoz produced no measurable changes in smooth muscle tone. Figure 7 illustrates the same point tested in a different way. A slow change in tension was produced in a ramp like waveform with a period of 40 sec. The threshold and peak discharge frequency were measured and a number of measurements were made at intervals in between. Data was obtained first at a P,, 2 of 40.6 mm Hg (circles) then at a Pcol of 12.5 mm Hg (crosses) and then at
I 50
I 25
0
TENSION Fig. 7. Response
of a single receptor
to slow ramp changes
40.6 mm Hg; crosses,
(9) in tension
12.5 mm Hg; triangles
with a period
37.3 mm Hg. Pco2.
of 40 sec. Circles,
312
‘3. W. BRADLEY
AND N. SC‘HEURMIER
6Or
1
I
0
Fig. 8. The effect of temperature Triangles
50
25
on discharge
TENSION (g) frequency. Slow ramp
changes
in tension
as in fig. 7
at 37 C; crosses at 30 C, and open circles at 38 C.
37.3 mm Hg (triangles). There was no change of discharge at the lower PcoL. Even though the tension changes were slow, a clear hysteresis effect was seen. At each level of tension the discharge frequency was higher when the tension was increasing.
In contrast to the lack of effect of changing COL, a fall in temperature depressed the discharge. This is shown in fig. 8 for the same receptor illustrated in fig. 7. Data measured at the lower temperature was obtained between the two higher temperatures. A similar response to a change of temperature was found in all 3 receptors subjected to this.
In three experiments 200 Llg of acetylcholine in solution were injected into the perfusion chamber in the region of the preparation. This produced contraction of the tracheal muscle with increase in tension. The discharge frequency of 5 receptors, seen as single-fibre activity, increased when acetylcholine was added and activity from several more receptors appeared. No case was seen in which discharge frequency effectively possibility
decreased with acetylcholine, which in series with the muscle. However, that some receptors are in parallel.
suggests that stretch receptors this does not entirely exclude
are the
Discussion The main advantage of studying receptor properties in vitro is that the effective stimulus to the receptor can be manipulated with greater precision than is possible in Go. The actual stimulus to which the tracheal stretch receptor responds is unknown although it is likely that they respond in some way to distortion of their
TRACHEAL
structure.
Tension
in the tracheal
STRETCH
RECEPTORS
itI vitro
wall is only an indirect
measure
373 of this distortion,
although it is more direct than measurement of the transtracheal pressure. Change in length of the preparation is unlikely to be a more direct measure of receptor distortion than tension. The relationship between receptor distortion and either length or tension changes would depend on the relative compliance and viscosity of the receptor and surrounding tissue. Direct measurement of airway wall tension has not been made in previous work on respiratory tract receptors, even with experiments in vitro (Taglietti and Casella, 1966; McKean, 1969; Bradley, 1974). Receptor distortion cannot be measured directly at the moment, and indeed the histology of these receptors is uncertain. Even their position in the wall of the trachea is in doubt. Bartlett et al. (1976a) believe the receptors to be in the muscle layer of dog’s trachea since receptors continued to discharge as pieces of tissue were removed until the muscle layer was reached. This would be consistent with the tindings of Larsell and Dow (1933) who demonstrated receptors in the vicinity of bronchial muscle in humans. On the other hand an electron microscopy study in rats (During et al., 1974) showed lanceolate receptor endings, believed to be stretch receptors, in bronchioles between the smooth muscle cell layer and the lamina propria. Similarly, a study using radioactively labelled bupivocaine in a rabbit (Bitenski rt al., 1975) suggested that the stretch receptors are close to the epithelium. Species differences, or differences in the relationship of tracheal and bronchial stretch receptors to their surrounding tissue, may be responsible for some of these apparently discrepant findings. The present study has shown that the receptors respond to an increase in tension produced by displacement in the transverse or longitudinal axis of the trachea, and that there is a dynamic component in the response. The response to changes in transverse tension has been quantified and shows a curvilinear relation with a threshold that varied considerably between receptors. The receptors studied were effectively in series with the muscle tibres because contraction
of the muscle,
induced
by acetyl-
choline, produced an increase in discharge frequency. These receptors are, therefore, similar in many respects to ‘in series tension receptors’ present in the alimentary tract (Iggo, 1955 ; Clarke and Davison, 1975). Changes in Pcoz did not alter the discharge characteristics
of these receptors, but a drop in temperature decreased discharge frequency as found for pulmonary stretch receptors in uiuo (Schoener and Frankel, 1972). The physiological importance of the effect of temperature in normal circumstances is unknown, but it is possible that the temperature of the tracheal mucosa differs during inspiration and expiration. The lack of response to a change in PC_ is surprising in view of the obvious increased sensitivity of pulmonary stretch receptors when the airway CO, falls below 20-30 mm Hg (Bradley et al., 1976). It would, however, be consistent with the findings of Bartlett and Sant’Ambrogio (1976) that bronchial and not tracheal stretch receptors are sensitive to a change in airway CO,. The receptor sites in the work of Bradley et al. were not localized, but it would be surprising, nevertheless, if all the receptors recorded from in their study were from bronchial stretch receptors, since about
G.
374
W.
BRADLEY
AND
N. SCHEURMIER
one-third of stretch receptors in the respiratory tract are in the trachea. Longitudinal pull on the trachea occurs in ciclo and it has been estimated that the resting force in dogs is approximately 50 g and a change from the supine to the head-up equivalent
position
produces
a tracheal
to 60 g (Miserocchi
lengthening
and Agostoni,
of 0.9 cm which is approximately 1973). This is due to the weight of
the heart and descent of the diaphragm. During inspiration there will be a further increase in longitudinal tension due to a downward pull from the diaphragm. Such changes in longitudinal tension are sufficient to cause tracheal stretch receptors to discharge and to alter the frequency of this discharge. In km, the pressure differential across the airway is used as a measure of the effective stimulus to these receptors. This is difficult to relate to changes in transverse tension in the airway wall because of the complex geometry of the airway. However, if Laplace’s law is applied to a trachea treated as a uniform cylinder and this is a gross simplification ~ and assuming a tracheal radius of I cm, then I cm Hz0 pressure differential is approximately equivalent to a tension of I g/cm; since the length of trachea used in this preparation was 9 cm. I cm H,O would be equivalent to 9 g wt applied to the preparation. In physiological conditions, the pressure swings that do occur in the extrathoracic trachea are small and in the opposite sense to the pressure changes in the intrathoracic trachea. During inspiration the pressure within the extrathoracic trachea becomes negative with respect to the pressure outside the trachea, but this could be consistent with an increase in discharge frequency since the tension in the posterior membrane will be increased as it is pulled into the tracheal lumen (see Bartlett ct 01. 1976a). However, the effect of longitudinal pull due to inspiration is probably greater. Changes in pressure during coughing are much larger and are likely to have very significant effects on discharge frequency. Whether this is of any physiological importance in the control of breathing during coughing is unknown. The intrathoracic trachea, on the other hand, is subjected to changes in intrapleural pressure during breathing which will have significant effects on transverse tension. The increase in longitudinal tension with inspiration, due to descent of the diaphragm. will add to the effect of these pressure changes. Large airways in the thorax are subjected to greater tension changes than smaller airways for three reasons. First, the radius of the larger airways is greater, and if Laplace’s law applies even qualitatively to the airways then a given pressure differential across a large airway will be associated with a greater tension than would the same pressure across a small airway (since T = PR for a cylinder, a large radius ~ R - for a given pressure differential - P - will produce a greater tension change ~ T). Secondly, during inspiration the pressure within an airway falls below atmospheric due to airflow resistance. This pressure drop will be greatest in the more peripheral airways, and consequently the pressure differential between intrapleural and airway pressure will be less in the smaller peripheral airways. Thirdly, it is known that the longitudinal pull on airways below the carina is small in dogs (Miserocchi and Agostoni, 1973). There may, therefore, be advantages in the siting of the majority
TRACHEAL
STRETCH
RECEPTORS
375
in UitrO
of stretch receptors in the trachea and main bronchi where tension changes are more marked, although the thickness of these airways may reduce the force applied to each receptor.
Heferences Adrian,
E. D.
J. Physiol. (London)
(1933).Afferent impulses in the vagus and their effect on respiration.
79: 332-358. Bartlett.
D., P. Jeffery. G. Sant’Ambrogio
trachea Bartlett,
D., G. Sant’Ambrogio J. Physiol.
receptors. Bartlett, Bitenski,
receptors
258
concerning
/London)
of the dog. Rrspir.
J. Chayen,
B. A. Cross,
the site of receptors
G. W., M. I. M. Noble and D. Trenchard
discharge
produced and its action
controlled
inflation
hypercapnia
on the discharge
the Hering-Breuer
(1975).
reflex. J. Physioi.
inflation
dioxide
J. Physiol.
and J. lravani
EnrnYckl.
concentration
(London)
261
of distension
J. Ph,siol.
: 427-429. stretch receptor
in dogs on cardio-pulmonary
: 359-373.
sensitive,
(London)
256:
(1974). The fine structure
vagal afferent
nerve endings
to
122Zl23P.
of the pulmonary
stretch
receptor
in
Gesch. 143: 215-222.
receptors
in the stomach
and the urinary
593-607. Larsell, 0. and R. S. Dow (1933). The innervation T. A. (1969). A linear approximation
of the frog. J. Appl. Physiol. G. and E. Agostoni
Scund. 91
(1976). The direct effect on pulmonary
(1975). Response
of the rat stomach.
Iggo. A. (1955). Tension
Miserocchi,
stretch
Phj,sio/. 26: 91-99.
in vitro. Acta Physiol.
recording
lung carbon
on breathing.
M., K. H. Andres
the rat. Z. Anal.
receptor
by changing
G. D. and J. S. Davison
McKean,
of tracheal
249: 30-3lP.
Bradley,
During,
properties
A. Guz, S. K. Jain and J. J. Johnstone
mediating
G. W. (1974). Pulmonary
bypass
in the
: 42 l-432.
Bradley,
Clarke,
of stretch receptors
258: 409420.
(1976). Effects of local and systemic
in the airways
L.. D. C. Chambers,
Evidence
(London/
and J. C. M. Wise (1976b). Transduction
(London)
D. and G. Sant’Ambrogio
of stretch
and J. C. M. Wise (1976a). Location
of the dog. .I. Physiol.
and bronchi
21: 775-78
of the human
of the transfer
bladder.
J. Physiol.
(London)
128:
lung. Am. J. Ana?. 52: 1255146.
function
of pulmonary
mechanoreceptors
I.
(1973). Longitudinal
forces acting
on the trachea.
Respir.
Physiol.
17:
62271. Miserocchi,
G., J. Mortola
in airways Schoener.
E. P. and H. M. Frankel
pulmonary Taglietti,
and G. Sant’Ambrogio
of dogs. J. Physiol. stretch
receptor.
V. and C. Casella
(London)
(1973). Localization
(1972). Effect of hyperthermia
Am. J. PhJsiol.
(1966). Stretch
of pulmonary
stretch
receptors
235: 775-782. and PaCO,
on the slowly adapting
222: 68-72.
receptors
stimulation
in frog’s
lungs.
Pjliigers
Arch.
292:
297-308. Widdicombe,
J. G. (1974).
Physiology, pp. 273-301.
Reflex
control
Series one. Vol. 2. edited
of breathing.
In: MTP
by J. G. Widdicombe.
International
Butterworths,
Review University
of Science. Park Press,