Respiration Physiology (1976) 21, 25%266;
North-Holland Publishing Company, Amsterdam
THE EFFECT OF LUNG REFLEXES ON THE PAlTERN OF BREATHING IN CATS’
A. J. WINNING’
and J. G. WIDDICOMBE
3 Department of Physiology, St. George’s Hospital Medical School, London SW17 OQT, and University Laboratory of Physiology, Oxford, United Kingdom
Abstract.
Tidal volume
anaesthetized injections during
of phenyl
eupnoea,
and at different
ditions. action
diguanide
hypercapnic tE.
The
stimulation
Recording
(tr) and expiratory type J (nociceptive)
and of histamine and hypoxic
VT/t1
relationship
during
have been measured
respectively.
receptors
for injection
The reflexes were studied
from hyperventilation
of the drugs was different
motor
was proportionally
tibres showed
in
by intravenous
the drugs caused rapid shallow breathing
from phrenic
of discharge
durations
rebreathing
but the tt/tE relationship
single unit activity
The results are interpreted
(tE)
and lung irritant
acid phosphate
hyperpnoea,
In all conditions
of breathing,
on the initial frequency
controls.
of alveolar
body temperatures.
tion in VT, tl and hypercapnic
(VT), and inspiratory
cats on stimulation
apnoea with reduc-
from that caused similar
that the lung reflexes
of the fibres, but cut short the discharge
earlier
in terms of the ways in which lung reflexes can modify
by
for all conhad little
than for the the pattern
of
breathing. Hyperpnoea
Pattern
Hyperthermia
Vagal reflexes
of breathing
Lung receptors
Extensive analysis has recently been made of the relationship between tidal volume (VT), inspiratory duration (tr) and expiratory duration (tE) during hypercapnia and hyperthermia in cats (Euler et al., 1970; Clark and Euler, 1972; Grunstein et al., 1973; Bradley et al., 1974a, b; Widdicombe and Winning, 1974). The nervous mechanisms underlying this control have been analysed, especially in relation to the Breuer-Hering inflation reflex and the organisation of the brainstem respiratory complex (Euler et al., 1973; Bradley et al., 1975). Apart from the Breuer-Hering inflation reflex, few other reflexes have been studied with respect to their action on the pattern of individual breaths. Karczewski (1975) using rabbits, has described the effect of some lung and respiratory tract reflexes, and his results suggest that the models of control of the pattern of breathing Accepred,for publicarion 9 April 1976.
’ A.W. was a MRC scholar. Research Council. ’ Present
address:
3 Present
address
University
The work was supported College
Hospital
of Dr. J. G. Widdicombe
by grants
Medical
and address 253
School,
from the Royal University
for correspondence.
Society and the Medical
St., London
WCI,
England.
254
A. _I. WINNING AND J. G. WIDDICOMBE
so far published may be over-simple. We have extended Karczewski’s work, using cats and a wider range of experimental conditions, and have obtained results different in some respects, which are described here. We have used phenyl diguanide as a stimulant of lung nociceptive (type I) receptors (Paintal, 1969), and histamine as a stimulant of lung irritant receptors (Mills et al., 1969) (see Discussion).
Methods
Sixteen adult cats were used with body weight 1.7 to 3.5 kg. The cats were injected intraperitoneally with 32 mg/kg of sodium pentobarbitone (Nembutal, Abbott). Supplementary doses were given intravenously when necessary to maintain surgical anaesthesia. Routine surgical preparation consisted of tracheal cannulation, the insertion of polyethylene catheters into a femoral artery and vein, and the placing of thread loops around both the cervical vagus nerves. Blood pressure was monitored from the arterial catheter using a strain-gauge manometer (Consolidated ElectroDynamics Corp.). An electrical heating blanket, thermostatically controlled from a thermocouple probe inserted into the rectum, was used to control body temperature. Additional heat was provided by an overhead infra-red lamp, when necessary. Body temperature was frequently checked by the use of a rectal mercury thermometer. Phrenic nerve activity was recorded from ‘single’ phrenic libres and from multitibre strands dissected out under oil in the neck from the cut uppermost root of the right phrenic nerve. Platinum electrodes and conventional amplifiers (Tektronix 122) were used. For records of integrated phrenic activity, the multi-libre signal was first rectified and then ‘integrated’, using a ‘leaky’ resistanceepacitance output (time constant 50 msec). Tidal volume was measured by integrating flow recorded from a Fleisch pneumotachograph head connected to the tracheal tube. Tidal CO2 % was measured by an infra-red absorption meter (Beckman-Spinco LBI), while arterial Po, and pH were determined by blood gas electrodes (Radiometer BMS 3). Most of the methods used have been described previously (Widdicombe and Winning, 1974). In the experiments involving rebreathing 0, from apnoea, the cats were first connected via the pneumotachograph head to a Palmer-Starling Ideal repiratory pump which hyperventilated them with intermittent positive pressure, until phasic phrenic discharge disappeared. In this apnoeic situation, a blood sample was taken for gas and pH analysis, after which the pump was switched off and the animal was connected to a 5 litre rebreathing bag initially tilled with pure 0,. Further blood samples were taken at the ends of rebreathing runs. The effects of lung reflexes in hypopnoeic cats were tested by injections in the hyperventilation apnoeic period before the resumption of phrenic discharge. Records of tidal volume, phrenic activity (single libre and integrated multi-Iibre)
LUNG REFLEXES AND PATTERN OF BREATHING
255
tidal CO, %, arterial blood pressure and an event marker were usually displayed on a Tektronix 551 oscilloscope and photographed on 7 cm paper using a modified Cosser camera. In a few experiments they were recorded on ultraviolet sensitive paper (Honeywell UV-31). In most experiments the signals were also stored on magnetic tape using a 7-channel recorder (Ampex SP-300), for subsequent analysis. Histamine acid phosphate (Evans Medical Ltd.), and phenyl diguanide hydrochloride (K and K Laboratories Inc.) were made up in 0.9 % saline. Doses are of the salts. Injections of 150 pg in 0.15 ml saline were used throughout, and made into the femoral venous catheter.
DEFINITIONS OF t1 AND tE, AND INTEGRATED
PHRENIC HEIGHT
tr is the interval between the initial increase and the start of the rapid decrease of integrated phrenic activity. tE is the interval between the end of one inspiratory duration and the beginning of the next, derived from the phrenic activity. ‘Integrated phrenic height’ is the peak amplitude of the integrated phrenic multitibre signal. The validity of the values for the inspiratory and expiratory durations of a breath as measured by the phrenic integrator system has been previously discussed by Widdicombe and Winning (1974). In VT/t1 plots some points have been omitted for clarity. All lines on graphs have been drawn by eye. Values related to augmented breaths have been omitted (e.g. fig. 1).
Results
THE VT/t1
RELATIONSHIP
Rebreathing 0, produces a near hyperbolic relationship between VT and tr, the limiting values of the two variables in a breath depending on the Breuer-Hering inflation reflex and the rate of build-up of phrenic motoneurone discharge (Clark and Euler, 1972). We have tested the effect of the two drugs on this relationship in a variety of conditions. Eupnoeic cats
Phenyl diguanide was given to sixteen cats. All exhibited rapid, shallow breathing usually occurring after an expiratory apnoea (fig. 1). These rapid, shallow breaths had smaller VT’Sand shorter tr’s than had the control breaths, as if the reflex-affected breaths had been ‘cut short’ before they reached the control values (fig. 2). In other words a line drawn through the control- and reflexly-influenced breaths runs close
A. J. WINNING AND J. G. W~DDICOMBE
256 Signal 203 ---B.P. (mm Hg 1 o Phrenic
I
1 5
set
Fig. I. Action of phenyl diguanide and histamine on phrenic single libre discharge. In each record (from above downwards) signal, blood pressure, tidal volume (zeroing at points of zero flow, inspiration upwards), and action potentials from single phrenic nerve libre. The top record shows the effect of 150 ng of phenyl diguanide injected into a femoral vein (lirst signal) via a catheter whose dead space was washed out with saline (second signal). There is expiratory apnoea followed by a short period of rapid, shallow breathing, with hypotension and bradycardia. The bottom record shows the effect of 150 pg of histamine acid phosphate. There is a short period of rapid, shallow breathing followed by an augmented breath, with hypotension and bradycardia.
oL 0
1.0
2.0
Fig. 2. The effect of I50 pg phenyl diguanide given intravenously to a eupnoeic cat whose vagus nerves were intact. The tidal volume of a breath (ordinate) is plotted against its inspiratory duration, tt (abscissa). x = control eupnoeic breaths; l = the first five breaths after the apnoea induced by phenyl diguanide. In this and subsequent figures lines have been drawn by eye to indicate possible relationships discussed in the text.
to the origin (fig. 2); the slope of this line indicates the average rate of increase of VT, and approximately of inspiratory drive (Clark and Euler, 1972). Changes in the integrated multi-fibre phrenic preparation height were used as an index of ‘total inspiratory centre drive’. With the a~inistration of phenyl diguanide, changes in the peak phrenic discharge were not always proportional to changes in VT, but with small doses of the drugs the proportionality was usually
251
LUNG REFLEXES AND PATTERN OF BREATHING
close. Sometimes the reduction in peak phrenic discharge was less than that of VT, probably because of the increased airway resistance due to bronchoconstriction (Karczewski and Widdicombe, 1969). In fifteen cats, histamine produced rapid shallow breathing and a Vr/tr relationship similar to that seen with phenyl diguanide, except that it was not preceded by an apnoeic period (fig. 1). Hyperpnoeic cats
The effect of lung reflexes during increased respiratory drive was investigated by making seven cats breathe an 8 o/0CO,-in-air mixture or an 8-12 o/00, in N, mixture. When a steady state had been reached, the effects of intravenous doses of phenyl diguanide or histamine were observed. Cats made hypercapnic or hypoxic showed breaths of greater VT and shorter tt than the normocapnic or normoxic control breaths, a result in accord with the analysis of the V-r/u relation by the rebreathing method (see Discussion). Phenyl diguanide or histamine in the seven cats reduced VT and tr along a line pointing towards the origin of the V-r/n relationship (fig. 3). 50
r I
10
“0”
40_
30.. 2
North-Holland Publishing Company, Amsterdam
THE EFFECT OF LUNG REFLEXES ON THE PAlTERN OF BREATHING IN CATS’
A. J. WINNING’
and J. G. WIDDICOMBE
3 Department of Physiology, St. George’s Hospital Medical School, London SW17 OQT, and University Laboratory of Physiology, Oxford, United Kingdom
Abstract.
Tidal volume
anaesthetized injections during
of phenyl
eupnoea,
and at different
ditions. action
diguanide
hypercapnic tE.
The
stimulation
Recording
(tr) and expiratory type J (nociceptive)
and of histamine and hypoxic
VT/t1
relationship
during
have been measured
respectively.
receptors
for injection
The reflexes were studied
from hyperventilation
of the drugs was different
motor
was proportionally
tibres showed
in
by intravenous
the drugs caused rapid shallow breathing
from phrenic
of discharge
durations
rebreathing
but the tt/tE relationship
single unit activity
The results are interpreted
(tE)
and lung irritant
acid phosphate
hyperpnoea,
In all conditions
of breathing,
on the initial frequency
controls.
of alveolar
body temperatures.
tion in VT, tl and hypercapnic
(VT), and inspiratory
cats on stimulation
apnoea with reduc-
from that caused similar
that the lung reflexes
of the fibres, but cut short the discharge
earlier
in terms of the ways in which lung reflexes can modify
by
for all conhad little
than for the the pattern
of
breathing. Hyperpnoea
Pattern
Hyperthermia
Vagal reflexes
of breathing
Lung receptors
Extensive analysis has recently been made of the relationship between tidal volume (VT), inspiratory duration (tr) and expiratory duration (tE) during hypercapnia and hyperthermia in cats (Euler et al., 1970; Clark and Euler, 1972; Grunstein et al., 1973; Bradley et al., 1974a, b; Widdicombe and Winning, 1974). The nervous mechanisms underlying this control have been analysed, especially in relation to the Breuer-Hering inflation reflex and the organisation of the brainstem respiratory complex (Euler et al., 1973; Bradley et al., 1975). Apart from the Breuer-Hering inflation reflex, few other reflexes have been studied with respect to their action on the pattern of individual breaths. Karczewski (1975) using rabbits, has described the effect of some lung and respiratory tract reflexes, and his results suggest that the models of control of the pattern of breathing Accepred,for publicarion 9 April 1976.
’ A.W. was a MRC scholar. Research Council. ’ Present
address:
3 Present
address
University
The work was supported College
Hospital
of Dr. J. G. Widdicombe
by grants
Medical
and address 253
School,
from the Royal University
for correspondence.
Society and the Medical
St., London
WCI,
England.
254
A. _I. WINNING AND J. G. WIDDICOMBE
so far published may be over-simple. We have extended Karczewski’s work, using cats and a wider range of experimental conditions, and have obtained results different in some respects, which are described here. We have used phenyl diguanide as a stimulant of lung nociceptive (type I) receptors (Paintal, 1969), and histamine as a stimulant of lung irritant receptors (Mills et al., 1969) (see Discussion).
Methods
Sixteen adult cats were used with body weight 1.7 to 3.5 kg. The cats were injected intraperitoneally with 32 mg/kg of sodium pentobarbitone (Nembutal, Abbott). Supplementary doses were given intravenously when necessary to maintain surgical anaesthesia. Routine surgical preparation consisted of tracheal cannulation, the insertion of polyethylene catheters into a femoral artery and vein, and the placing of thread loops around both the cervical vagus nerves. Blood pressure was monitored from the arterial catheter using a strain-gauge manometer (Consolidated ElectroDynamics Corp.). An electrical heating blanket, thermostatically controlled from a thermocouple probe inserted into the rectum, was used to control body temperature. Additional heat was provided by an overhead infra-red lamp, when necessary. Body temperature was frequently checked by the use of a rectal mercury thermometer. Phrenic nerve activity was recorded from ‘single’ phrenic libres and from multitibre strands dissected out under oil in the neck from the cut uppermost root of the right phrenic nerve. Platinum electrodes and conventional amplifiers (Tektronix 122) were used. For records of integrated phrenic activity, the multi-libre signal was first rectified and then ‘integrated’, using a ‘leaky’ resistanceepacitance output (time constant 50 msec). Tidal volume was measured by integrating flow recorded from a Fleisch pneumotachograph head connected to the tracheal tube. Tidal CO2 % was measured by an infra-red absorption meter (Beckman-Spinco LBI), while arterial Po, and pH were determined by blood gas electrodes (Radiometer BMS 3). Most of the methods used have been described previously (Widdicombe and Winning, 1974). In the experiments involving rebreathing 0, from apnoea, the cats were first connected via the pneumotachograph head to a Palmer-Starling Ideal repiratory pump which hyperventilated them with intermittent positive pressure, until phasic phrenic discharge disappeared. In this apnoeic situation, a blood sample was taken for gas and pH analysis, after which the pump was switched off and the animal was connected to a 5 litre rebreathing bag initially tilled with pure 0,. Further blood samples were taken at the ends of rebreathing runs. The effects of lung reflexes in hypopnoeic cats were tested by injections in the hyperventilation apnoeic period before the resumption of phrenic discharge. Records of tidal volume, phrenic activity (single libre and integrated multi-Iibre)
LUNG REFLEXES AND PATTERN OF BREATHING
255
tidal CO, %, arterial blood pressure and an event marker were usually displayed on a Tektronix 551 oscilloscope and photographed on 7 cm paper using a modified Cosser camera. In a few experiments they were recorded on ultraviolet sensitive paper (Honeywell UV-31). In most experiments the signals were also stored on magnetic tape using a 7-channel recorder (Ampex SP-300), for subsequent analysis. Histamine acid phosphate (Evans Medical Ltd.), and phenyl diguanide hydrochloride (K and K Laboratories Inc.) were made up in 0.9 % saline. Doses are of the salts. Injections of 150 pg in 0.15 ml saline were used throughout, and made into the femoral venous catheter.
DEFINITIONS OF t1 AND tE, AND INTEGRATED
PHRENIC HEIGHT
tr is the interval between the initial increase and the start of the rapid decrease of integrated phrenic activity. tE is the interval between the end of one inspiratory duration and the beginning of the next, derived from the phrenic activity. ‘Integrated phrenic height’ is the peak amplitude of the integrated phrenic multitibre signal. The validity of the values for the inspiratory and expiratory durations of a breath as measured by the phrenic integrator system has been previously discussed by Widdicombe and Winning (1974). In VT/t1 plots some points have been omitted for clarity. All lines on graphs have been drawn by eye. Values related to augmented breaths have been omitted (e.g. fig. 1).
Results
THE VT/t1
RELATIONSHIP
Rebreathing 0, produces a near hyperbolic relationship between VT and tr, the limiting values of the two variables in a breath depending on the Breuer-Hering inflation reflex and the rate of build-up of phrenic motoneurone discharge (Clark and Euler, 1972). We have tested the effect of the two drugs on this relationship in a variety of conditions. Eupnoeic cats
Phenyl diguanide was given to sixteen cats. All exhibited rapid, shallow breathing usually occurring after an expiratory apnoea (fig. 1). These rapid, shallow breaths had smaller VT’Sand shorter tr’s than had the control breaths, as if the reflex-affected breaths had been ‘cut short’ before they reached the control values (fig. 2). In other words a line drawn through the control- and reflexly-influenced breaths runs close
A. J. WINNING AND J. G. W~DDICOMBE
256 Signal 203 ---B.P. (mm Hg 1 o Phrenic
I
1 5
set
Fig. I. Action of phenyl diguanide and histamine on phrenic single libre discharge. In each record (from above downwards) signal, blood pressure, tidal volume (zeroing at points of zero flow, inspiration upwards), and action potentials from single phrenic nerve libre. The top record shows the effect of 150 ng of phenyl diguanide injected into a femoral vein (lirst signal) via a catheter whose dead space was washed out with saline (second signal). There is expiratory apnoea followed by a short period of rapid, shallow breathing, with hypotension and bradycardia. The bottom record shows the effect of 150 pg of histamine acid phosphate. There is a short period of rapid, shallow breathing followed by an augmented breath, with hypotension and bradycardia.
oL 0
1.0
2.0
Fig. 2. The effect of I50 pg phenyl diguanide given intravenously to a eupnoeic cat whose vagus nerves were intact. The tidal volume of a breath (ordinate) is plotted against its inspiratory duration, tt (abscissa). x = control eupnoeic breaths; l = the first five breaths after the apnoea induced by phenyl diguanide. In this and subsequent figures lines have been drawn by eye to indicate possible relationships discussed in the text.
to the origin (fig. 2); the slope of this line indicates the average rate of increase of VT, and approximately of inspiratory drive (Clark and Euler, 1972). Changes in the integrated multi-fibre phrenic preparation height were used as an index of ‘total inspiratory centre drive’. With the a~inistration of phenyl diguanide, changes in the peak phrenic discharge were not always proportional to changes in VT, but with small doses of the drugs the proportionality was usually
251
LUNG REFLEXES AND PATTERN OF BREATHING
close. Sometimes the reduction in peak phrenic discharge was less than that of VT, probably because of the increased airway resistance due to bronchoconstriction (Karczewski and Widdicombe, 1969). In fifteen cats, histamine produced rapid shallow breathing and a Vr/tr relationship similar to that seen with phenyl diguanide, except that it was not preceded by an apnoeic period (fig. 1). Hyperpnoeic cats
The effect of lung reflexes during increased respiratory drive was investigated by making seven cats breathe an 8 o/0CO,-in-air mixture or an 8-12 o/00, in N, mixture. When a steady state had been reached, the effects of intravenous doses of phenyl diguanide or histamine were observed. Cats made hypercapnic or hypoxic showed breaths of greater VT and shorter tt than the normocapnic or normoxic control breaths, a result in accord with the analysis of the V-r/u relation by the rebreathing method (see Discussion). Phenyl diguanide or histamine in the seven cats reduced VT and tr along a line pointing towards the origin of the V-r/n relationship (fig. 3). 50
r I
10
“0”
40_
30.. 2
