The transduction properties of tracheal stretch receptors in vitro

The transduction properties of tracheal stretch receptors in vitro

Re.\p;rurion 0 t’/~r.sio/o~~~ ( 1977) 31, 365 -375 El.sevierNorth-Holland Biomedical Press THE TRANSDUCTION PROPERTIES OF TRACHEAL, STRETCH KECEP...

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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.

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E. D.

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(1933).Afferent impulses in the vagus and their effect on respiration.

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the Hering-Breuer

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(1974). The fine structure

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(1976). The direct effect on pulmonary

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