inhibition of arterial baroreceptor and chemoreceptor reflex responses by neuropeptide y in...

Journal of the Autonomic Nervous System, 39 (1992) 169-180 169 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00

JANS 01287

Inhibition of arterial baroreceptor and chemoreceptor reflex responses by neuropeptide Y in anaesthetised dogs

Mark Moriarty, Erica K. Potter and D.I. McCloskey Prince of Wales Medical Research Institute, c / o School of Physiology and Pharmacology, University of New South Wales,

Sydney, Australia

(Received 16 May 1991) (Revision received 20 March 1992)

(Accepted 27 March 1992)

Key words: B a r o r e c e p t o r ref lex; C h e m o r e c e p t o r ref lex; N e u r o p e p t i d e Y; V a g u s

Abstract

The effects of bolus intravenous injections of neuropeptide Y (NPY) on increases in pulse interval (PI) evoked reflexly by arterial chemoreceptor and baroreceptor stimulation were investigated in anaesthetised dogs. The arterial chemoreceptors were stimulated by rapid injections of small volumes of CO 2 into the carotid sinus or brief episodes of tracheal occlusion. Intravenous injections of NPY produced a prolonged attenuation of the reflex prolongation of PI induced by both methods. Two methods of testing the arterial baroreceptor reflex were used: steady-state increases in PI evoked in response to maintained step increases in systolic arterial blood pressure (SABP) from inflation of an aortic balloon-tipped catheter, and beat-by-beat increases in PI evoked reflexly by 'ramp' increases in blood pressure caused by intravenous injections of phenylephrine. In both methods the relationship between SABP and PI is linear over the range tested (up to SABP 200 mmHg), the slope of the line indicating the sensitivity of the reflex response. Intravenous injections of NPY produced a prolonged attenuation of the baroreceptor-cardiodepressor reflex measured by both methods. No significant differences were observed between the NPY-mediated inhibition of the direct effects on PI of electrical stimulation of a vagus nerve, and its inhibition of the reflex responses of PI to chemoreceptor or baroreceptor stimulation. The results indicate that the attenuation of reflex PI responses to arterial chemoreceptor and baroreceptor stimulation following an intravenous injection of NPY can be accouiated for in terms of the action of NPY on vagal nerve endings at the heart, although additional sites of action cannot be ruled out.

Introduction

N e u r o p e p t i d e Y ( N P Y ) is a 36 a m i n o ac id

p e p t i d e wh ich was f irs t e x t r a c t e d f r o m p o r c i n e

b r a i n [35,36]. T h e p a t t e r n o f d i s t r i bu t i on o f N P Y

Correspondence to: E.K. Potter, Prince of Wales Medical Research Institute, c /o School of Physiology and Pharmacol- ogy, University of New South Wales, P.O. Box 1, Kensington, Sydney, NSW 2033, Australia.

in t h e c a r d i o v a s c u l a r sys tem a p p e a r s to be o n e o f

co loca l i s a t i on wi th n o r a d r e n a l i n e in s y m p a t h e t i c

n e r v e f ib res to b l o o d vesse l s and t h e h e a r t

[1,6,10,16]. F o l l o w i n g a p e r i o d o f c a r d i a c sympa-

t he t i c n e r v e s t i m u l a t i o n in a n a e s t h e t i s e d dogs ,

t h e r e is a p r o l o n g e d a t t e n u a t i o n o f t h e s lowing of

h e a r t r a t e p r o d u c e d by s t i m u l a t i o n o f t he vagus

n e r v e [25,26,40]. Th i s i nh ib i t i on o f vaga l ac t ion at

t he h e a r t is r e s i s t an t to a - a n d / 3 - a d r e n o c e p t o r

b l o c k a d e , and is n o t m i m i c k e d by bo lus in t ra -

v e n o u s in j ec t ions o f n o r a d r e n a l i n e [25]. H o w e v e r ,

bo lus i n t r a v e n o u s in j ec t ions o f N P Y do r e p r o -

duce this prolonged inhibition of cardiac vagal action. It has been proposed, therefore, that NPY or an NPY-like substance released by sympathetic stimulation is responsible fl~r this inhibition [25]. The action is a presynaptic one, on postgan- glionic vagus nerve fibres [26].

On the basis of this vagal inhibitory action of NPY it can be predicted that NPY will also influence reflex adjustments in heart rate. Two major reflexes which activate vagal efferent fibres to slow the heart are the arterial chemoreceptor and baroreceptor reflexes. A fall in the oxygen tension of arterial blood stimulates arterial chemoreceptors, which in turn reflexly activate respiratory activity and vagal cardioinhibitory fibres [5]. When the arterial baroreceptors are stimulated by an increase in arterial blood pressure, reflex excitation of vagal cardioinhibitory fibres and inhibition of sympathetic cardiac nerves evoke a decrease in heart rate. The vagally-mediated contribution is prominent in the initial stages of responses to rapid increases in pressure [23,37]. If NPY acts only on vagal nerve terminals, then it can be predicted that NPY would attenuate reflex bradycardia evoked by arterial chemoreceptor or baroreceptor stimulation. Furthermore, the direct inhibition of cardiac vagal action by NPY and the inhibition of reflexly induced bradycardia would be expected to be of similar magnitude and duration. Any discrepancies between these two effects could provide evidence of a further role of NPY acting elsewhere along the reflex pathways, for example, by modifying the responsiveness of chemoreceptor [27] or baroreceptor nerve endings, or the transmission through central neural pathways.

In the present study the inhibitory action of NPY on heart rate responses evoked reflexly and by electrical stimulation of the cervical vagus nerve were compared in anaesthetised dogs. To accomplish this, methods for obtaining reproducible changes in heart rate by arterial chemoreceptor and baroreceptor stimulation were em- ployed. These tests had to be of short duration with a quick recovery time, to allow for repeated testing before and after NPY administration. A new test for the chemoreceptor-cardiodepressor reflex was developed to meet these requirements.

Materials and Methods

Twenty-four mongrel dogs of both sexes, weight range 3-10 kg, were used. The animals were anaesthetised initially with intravenous thiopen- tone (15 mg/kg , Pentothal, Abbott), followed by intravenous a-chloralose (50-10 mg/kg , Sigma). A femoral vein was cannulated for drug administration and supplements of chloralose. The trachea was cannulated low in the neck. The animals breathed spontaneously. Airway pressure was measured during experimental tracheal occlusions via a small nylon tube (internal diameter of 3 ram) inserted down the tracheal cannula, and connected to a Grass volumetric pressure transducer (PT5A). This was recorded on one channel of a Grass polygraph. A femoral artery was cannulated for continuous blood pressure recording via a Gould Statham pressure transducer (P23AC) connected to a second channel of the polygraph. The electrocardiogram was recorded through subcutaneous needle electrodes and displayed on a storage oscilloscope. The electrocardiogram was used to obtain beat-by-beat pulse interval (PI), after processing with Neurolog modules (Dig- itimer, UK). Deep abdominal temperature was maintained in the range_<37-38°C.

Experimental protocol The basic experimental protocol used was to

obtain a series of reproducible control responses in PI to arterial chemoreceptor stimulation, baroreceptor stimulation and electrical stimulation of the left vagus nerve. The animal was then given a bolus intravenous injection of NPY (30-60 /zg/kg, 7-14 nmol /kg , human, Pacific Biotech- nology, Sydney) and alternate stimulation of the reflexes and vagus nerve was continued at regular intervals until any change in the reflex PI responses had returned to control levels.

Studies have shown the predominance of the right vagus nerve over the left in controlling heart rate [22], so the right vagus nerve was left intact to assure good PI responses to reflex stimulation. The left vagus nerve was isolated in the lower cervical region, cut and laid across platinum electrodes for stimulation. Supramaximal stimuli ( 517 V, 1 ms) were delivered to the left vagus in

trains of 5-s duration, at intervals of 30 s. The frequency of vagal stimulation was set between 2 and 8 Hz to give a submaximal increase in PI, between 150-250 ms. In some animals this experiment was performed after effective /3-blockade with propranolol (1.5 mg/kg, Inderal, ICI).

Arterial chemoreceptor reflex stimulation A standard method of stimulating the arterial

chemoreceptors was initially used [11]. Brief chemoreceptor stimuli were delivered into the carotid sinus region by the sudden retrograde injections via a cannulated external carotid artery of small volumes (0.1-0.5 ml) of warmed, physiological saline through which CO 2 had been bub- bled. This method evoked brief, but substantial increases in PI. However, these reflex increases in PI were typically variable. Also, in some in- stances saturation of the reflex response was thought to have occurred because larger stimuli failed to increase it.

Thus, an alternative method was developed for stimulating the arterial chemoreceptors. This also had to meet the requirement of being short in duration of effect to enable its repeated applica- tion. It was adapted from a procedure developed by Callanan and Read [3]. In anaesthetised rabbits, they found that during tracheal occlusion for five consecutive breaths there was a progressive augmentation of inspiratory effort in each occluded breath. They concluded that the augmentation of inspiratory effort depended significantly upon activation of the arterial chemoreceptors, as it was significantly reduced after the animal had been given oxygen to breathe for 5 min immediately prior to occlusion and after carotid denerva- tion.

In the similar experiments reported here on dogs it was noted that during tracheal occlusion for five consecutive breaths there was a prolongation of PI following each obstructed inspiratory effort. The mechanism underlying these responses in PI during tracheal occlusion was investigated in eight dogs, in which control tracheal occlusions for five consecutive breaths were performed. A recovery time of at least 2 min was allowed between each tracheal occlusion. The animal was then given oxygen to breathe for 5

171

min, and the trachea was then immediately occluded again for five breaths. In five of the eight dogs studied in these tests, arterial blood was taken at the beginning of the fifth occluded breath, for both control and test occlusions, for analysis of pH, Pco: and Po2 (Micro 13 Analyzer: Instrumentation Laboratories).

Arterial baroreceptor reflex stimulation Two methods of stimulation were used for the

arterial baroreceptor reflex. The first method used to raise blood pressure was a balloon tip catheter positioned in the abdominal aorta to obtain steady-state responses. In the dogs used in these tests blood pressure was recorded via a cannulated lingual artery, beyond the carotid sinus baroreceptors, rather than a femoral artery. Sys- tolic arterial blood pressure (SABP) was maintained at a series of stepwise levels (generally 10-15 mmHg steps) up to a maximum SABP level of no more than 200 mmHg. Each pressure level was maintained until the PI response had reached a new steady-state. This took between 10 and 15 s. This was done for several different pressure levels in each test. SABP was then plotted against the steady-state PI response for each test, a linear regression function was fitted, and the slope of this line was taken as a measure of the reflex sensitivity for that particular test. For all tests, the linear relationship between SABP and PI was found to be an adequate fit [42], with coefficient of determination (R 2) values of 0.76 or greater (mean 0.90, range 0.76-0.99).

The second method was based on the ' ramp' method, which relates the change in PI to an increase in SABP evoked by an intravenous injection of phenylephrine [33]. The relationship between beat-by-beat SABP and those pulse intervals which begin with the next beat is a linear one [34]. The slope of this line indicates the reflex sensitivity. In the present study intravenous injections of phenylephrine (15-100/zg) were used to raise SABP. This method has been directly vali- dated by baroreceptor afferent and cardiac vagal efferent nerve recordings in anaesthetised dogs [15]. Beat-by-beat SABP was plotted against the PI beginning with the next beat, and a linear regression function fitted to these points. The

172

slope of this line was taken as the measure of reflex sensitivity for each individual test. Again, the range of blood pressures tested was from the resting SABP up to a maximum of no more than 200 mmHg, and the SABP-PI relation is regarded as approximately linear over this range [34]--although, of course, it is known to be markedly sigmoid when taken over a complete range from well below control levels to levels higher than those used here [14]--with a mean R 2 value of 0.87 (range 0.60-0.99).

Both steady-state and phasic baroreceptor stimulations were found to be quick and reproducible methods of evoking reflex PI responses. No new test was initiated until SABP and PI had returned fully to control levels following the previous test. Usually this took less than 2 min and enabled regular testing over extended periods. In both steady-state and phasic tests, points which fell during the inspiratory phase of the respiratory cycle were discarded because the vagal cardiodepressor reflexes are at tenuated at this time [11,34].

Data analysis In order to compare the NPY-mediated inhibi-

tion of the whole arterial chemoreceptor and baroreceptor reflexes with the inhibition simply of cardiac vagal action, the maximum percent inhibition (MPI) and the time to half recovery (T50) for the responses were calculated. The MPI for the NPY-mediated inhibition of cardiac vagal action was calculated as follows: control stimuli were delivered to the left vagus to obtain reproducible increases in pulse interval (API). Then, after an intravenous NPY injection, intermittent stimulation of the left vagus nerve was continued until API had returned to control levels. The timecourse of recovery of NPY-mediated inhibition of cardiac vagal action has been shown to be best described by a linear function [8]. The AP1 values following intervention were plotted against time, and a regression line calculated. The API at its point of maximal inhibition was calculated from this regression line, at 2 min after NPY injection. This time coincides with the most fre- quently observed time of maximum inhibition. The use of the regression equation to calculate the maximally inhibited value of API was pre-

ferred to simply recording the smallest APi observed, because the regression equation made the figure recorded dependent upon the full series of points observed after NPY rather than one single observed point. MPI was calculated using the equation:

API c - ApI t MPI - x 100

API~

where API c and ApI, are the control and treat- ment API responses respectively. The T50 was also calculated from the regression line. A similar method was used for calculating the MPI and T50 for the NPY-mediated inhibition of the reflex responses. For the chemoreceptor reflex, the sum of the increases in PI for each of the five occluded breaths (SUM API, see Results for ex- planation) for each occlusion test following NPY injection were plotted against time, and a regression line calculated. The maximally inhibited SUM API was calculated from this regression line, at 2 min after NPY injection. This maximally inhibited SUM API was compared with control SUM API to give MPI. The T50 was also calculated from the regression line. In the case of the baroreceptor reflex, the slopes of the SABP against PI graphs for each test following NPY injection were plotted against time, and a regression line calculated. The maximally inhibited reflex response was calculated from this regression line, at 2 min after NPY injection. This maximally inhibited reflex response was compared with control responses to give MPI. The T50 was also calculated from the regression line.

Paired t-tests were used to compare the MP1 and T50 of the inhibition of the reflex response with that of cardiac vagal action, in order to account for any variability between animals of these responses. All results for MPI and T50 are presented as the mean _+ standard error of the mean.

Results

Chemoreceptor reflex testing by tracheal occlusion During tracheal occlusion in the dog there is a

marked increase in PI following each occluded

P.[. (ms)

1200

4 0 0

A,P. (mmHg)

- 3 0

P.k (ms)

1200

400

After 5min. (3=

A.P.

(mmHg)

- 3 0

I I 20 sac

Fig. 1. Recordings from an anaesthetised dog to illustrate the response of pulse interval during brief tracheal occlusion before and after breathing oxygen. The top two panels show pulse interval (PI) and airway pressure (AP) records taken during occlusion of the trachea for five consecutive breaths. Note that there is a marked increase in PI following each [nspiratory effort during occlusion. There is also augmentation of inspiratory effort during occlusion. The bottom panel is taken during a similar tracheal occlusion, immediately following a period of 5 min of breathing oxygen. During the period of occlusion there is less augmentation of the increase

in PI after each inspiratory effort.

inspiratory effort (see Fig. 1). When the trachea is occluded for five consecutive breaths, progressive augmentat ion of these increases in PI occurs. In eight dogs the responses of PI during tracheal occlusion were tested immediately after inspiring oxygen for 5 min. Following oxygen, the augmentation of the increase in PI during tracheal occlusion was significantly reduced, as shown for example in the records reproduced in Fig. 1. For each of the eight dogs tested, the increase in PI for each of the five occluded breaths after oxygen was found to be significantly less than the aver- aged increase in PI for each of the five occluded breaths in control responses recorded while the

173

animals brea thed air (paired-sample t-test, P < 0.05). The reduction in this response for each breath following oxygen ranged from 45% to 59%. This element of the response was attributed solely to arterial chemoreceptors. For this reason, the sum of the increase in PI for all five occluded breaths (SUM API) was taken as the measure of arterial chemoreceptor reflex sensitivity.

In five dogs arterial blood samples were taken at the beginning of the fifth occluded breath. During control tracheal occlusions, Po2 levels were 39 + 3 m m H g (range 30-47 mmHg). After 5 min of oxygen inspiration, arterial Po2 was significantly higher at the fifth occluded breath (paired-sample t-test, P < 0.005) during tracheal occlusion at 340 + 16 m m H g (range 300-390 mmHg). No difference in arterial Pco2 during tracheal occlusion before (43 + 5 mmHg, range 35-55 mmHg) and after (53 + 9 mmHg, range 36-80 mmHg) oxygen could be demonstrated sta- tistically. However, there was a significant decrease in arterial pH from 7.30 + 0.04 (range 7.20-7.37) to 7.23 + 0.0.04 (range 7.12-7.33) after oxygen (paired-sample t-test, P < 0.005).

Effect of NPY on the arterial baroreceptor and chemoreceptor reflexes

Initial experiments showed that the reflex increase in PI to chemoreceptor stimulation by retrograde intracarotid injections of small volumes of physiological saline equilibriated with CO 2 were markedly reduced, and in one case completely abolished (see Fig. 2), following a bolus intravenous injection of NPY. However, this reflex response was typically variable, even in control conditions, and did not lend itself to the kind of quantitative analysis required in the present study. Therefore, tracheal occlusion for five consecutive breaths was adopted as a means of stimulating the arterial chemoreceptor reflex.

In nine dogs the NPY-mediated inhibition of the arterial chemoreceptor reflex was compared with that of cardiac vagal action. The results for these experiments are summarised in Table I. Following an intravenous injection of NPY (30-60 /zg/kg), the increase in PI in response to tracheal occlusion was significantly reduced. When the inhibition of the effects of electrical stimulation

174

CONTROL 800 [ PI

(ms) ~ k~ ] , ~ ~_ L. [ 250

• • • • • • •

BP 2 0 0

Cm " 'tO o [ lmin

L I

A F T E R N P Y

I (ms) 2 5 0 . . . .

BP 2 0 0 ~-

(mmHg)lO 0 L 1 7 rains

Fig. 2. Recordings of pulse interval (PI) and arterial blood pressure (BP) from an anaesthetised dog to demonstrate the effect of NPY on the arterial chemoreceptor reflex response. Illustrated are the PI responses to arterial chemoreceptor stimulation (*) by retrograde injections of small volumes of CO 2 equilibriated physiological saline into the carotid sinus. The bottom panel is taken after a bolus intravenous injection of NPY (44 /xg/kg), and shows the PI response to chemoreceptor stimulation is significantly reduced for a prolonged period. The swings in the arterial pressure trace that follow each chemoreceptor stimulus are secondary to the large breaths evoked by these stimuli--note that after NPY, these still continue, indicating that the chemoreceptor stimuli were effectively transmitted to the central nervous system, although

they failed to evoke bradycardia.

o f the vagus ne rve was c o m p a r e d with the inhibi -

t ion o f the re f lex r e s p o n s e , no s igni f icant d i f fer -

e n c e in e i t h e r t he m a g n i t u d e o r t i m e - c o u r s e cou ld

be d e m o n s t r a t e d (pa i r ed t- test , T a b l e I). Th i s was

also t he case for e x p e r i m e n t s p e r f o r m e d a f t e r

e f fec t ive / ~ - a d r e n o c e p t o r b l o c k a d e with p r o p r a -

nolol .

F o l l o w i n g an i n t r a v e n o u s in j ec t ion o f N P Y the

inc rease in PI in r e s p o n s e to a r te r i a l b a r o r c c e p -

tor s t imu la t i on ( s t eady - s t a t e m e t h o d ) was signifi-

cant ly r e d u c e d . W h e n the inh ib i t ion o f the e f fec t s

o f e l ec t r i ca l s t imu la t i on o f the vagus ne rve was

c o m p a r e d wi th t he inh ib i t ion o f t he re f lex re-

sponse , no s ign i f i can t d i f f e r e n c e in e i t h e r the

m a g n i t u d e o r t i m e - c o u r s e cou ld be d e m o n s t r a t e d

(pa i r ed t- test , T a b l e 1).

In fou r dogs, b a r o r e c e p t o r re f lex s t i m u l a t i o n

( ' r a m p ' m e t h o d ) was p e r f o r m e d , t o g e t h e r wi th

s t a n d a r d e lec t r i ca l s t i m u l a t i o n of the cut vagus

nerve , in t he s a m e e x p e r i m e n t a l run, b e f o r e and

a f t e r i n t r a v e n o u s in jec t ion o f a bo lus dose o f

N P Y . Th i s e x p e r i m e n t was p e r f o r m e d b e f o r e (n

= 4) and a f te r (n = 3) e f f ec t ive / 3 - a d r e n o c e p t o r

b l o c k a d e wi th p r o p r a n o l o l (1.5 m g / k g ) . N P Y

e v o k e d a s ign i f i can t and p r o l o n g e d inh ib i t ion o f

t he b a r o r e c e p t o r re f lex r e s p o n s e and of c a rd i ac

vagal ac t ion . W h e n the M P I for t h e inh ib i t ion o f

the re f lex r e s p o n s e and vagus n e r v e r e s p o n s e was

TABLE I

Comparison of NPY-mediated inhibition of arterial baroreceptor and chemoreceptor reflex responses with inhibition of cardiac z'agal action

Chemoreceptor Vagus Steady-state Vagus Ramp Vagus reflex baroreceptor baroreceptor

reflex reflex

n = 9 MPI (%) 63 -+ 7 64 .+ 7 n.s. T50(min) 33+ 6 42+_5n.s.

Propanolol n = 5 MPIP (%) 93 -+ 13 58 + 8 n.s. T50 p(min) 51+ 9 41 +5n.s.

4 4 46.+10 55+ 9n.s. 53+11 63+ 6n.s. 21+ 6 35_+ 10n.s. 22-+ 4 37_+ 4 *

3 76 + 15 61 _+ 12 n.s. 31+ 12 39+ 3n.s.

e indicates experiments performed after propanolol (1.5 mg/kg). n.s. no significant difference. * P < 0.05 for significance of reflex responses compared with vagus responses (paired t-test).

175

D 0 , .

200"

tO0'

-~o ~ 2~ ,~ 6b 8~

• , , , • , ,

-20 20 40 6 0 8 0

t2°° 1 i °

1 . . . . . . . . , . i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4001 " ,

0 -2o o 20 40 so 80

TI RE (mi n) Fig. 3. Results from an anaesthetised dog all obtained during the same experimental run, and showing the inhibitory action of an intravenous injection of NPY (40 ~g/kg) on the reflex responses of pulse interval to stimulations of the arterial chemoreceptors and baroreceptors, and a comparison of these with the effects on pulse interval of standard trains of electrical stimuli applied to the cut left vagus nerve. This experiment was performed after effective /3-adrenoceptor blockade with propranolol (1.5 mg/kg). In the top graph each point shows the change in PI (dPl) produced by a standard electrical stimulation of the left vagus nerve. In the middle graph each point represents the slope of the PI and SABP relationship. In the bottom graph each point shows the sum of the increases in PI (SUM API) for five occluded inspiratory efforts. The injection of NPY was given at time = 0. This resulted in a prolonged inhibition of all three responses. Linear regression functions were fitted to points beginning 2 min after NPY injection, and from these lines MPI and T50 were calculated. The MPIs were 63%, 100% and 87% for the vagal, baroreceptor reflex and chemoreceptor reflex responses respectively,

whilst the T50 values were 44, 22 and 39 min respectively.

compared, no significant difference could be demonstrated (paired t-test, Table I). However, the T50 of the vagal inhibition was significantly larger (paired t-test, P < 0.05). No significant difference in either MPI or T50 of the reflex re-

sponse and vagus nerve response could be demonstrated after propranolol.

Figure 3 is an example of the results from one experiment, performed in the presence of /3- adrenoceptor blockade with propranoiol, showing the NPY-mediated inhibition of PI responses to tracheal occlusion, arterial baroreceptor stimulation ('ramp' method) and electrical stimulation of the vagus nerve.

Discussion

It has been proposed that NPY released on cardiac sympathetic nerve stimulation has a prolonged prejunctional inhibitory action on the vagus nerve at the heart [25,26,40]. It has been shown in man that upon sympathetic activation there is an increase in the plasma level of NPY [17,21]. In the pig and in the dog, direct stimulation of the cardiac sympathetic nerves leads to an increase in the overflow of NPY-like immunoreactive material into the coronary sinus [29,41].

This inhibitory action of NPY on cardiac vagal action would be expected to play an important role in modifying reflex adjustments in heart rate. We examined two important reflexes here, the arterial chemoreceptor and baroreceptor refexes. A new method, adapted from an established test for chemoreceptor stimulation of inspiratory efforts [3], is described here for stimulating arterial chemoreceptors and monitoring changes in reflexly evoked increases in pulse interval. A more orthodox method of injecting small volumes of CO 2 equilibriated physiological saline into the carotid sinus region [11] was also used in initial experiments. This confirmed that NPY attenuated the chemoreceptor reflex action on heart rate, but the method was unsatisfactory for this study because of the intrinsic variability of responses evoked using it. The responses of PI to tracheal occlusion in anaesthetised dogs reported here proved to be a quick and reproducible method for analysis of changes in arterial chemoreceptor reflex activity over time. The increase in PI for each of the five occluded breaths was significantly reduced following oxygen. The part of the response reduced in this way was

1 7~'+

attributed to stimulation of the arterial chemoreceptors. Some increase in PI did remain following oxygen. This could perhaps be the result of stimulation of laryngeal receptors [2] or, in its very late stages, of central chemoreceptors [3].

A bolus intravenous injection of NPY was found to produce a prolonged attenuation of the increases in PI evoked during tracheal occlusion. In experiments where the NPY-mediated inhibition of the chemoreceptor reflex response was compared with that of the response to electrical stimulation of the vagus, it was found that there was no significant difference in either the MPI or the T50. This was also the case after effective ~-adrenoceptor blockade with propranolol. Thus, while other sites of action cannot be ruled out, the results reported here provided no evidence that NPY, at the arterial concentrations achieved after intravenous bolus doses of the magnitudes used here, acted elsewhere in the chemoreceptor to vagal cardiodepressor reflex pathway than at its well-documented site of action on the cardiac vagal nerve terminals [25,26,40]. Previous experiments have shown NPY-like immunoreactivity in sympathetic nerve fibres to the smooth muscle cells of blood vessels in the carotid body of the rat [13]. Furthermore, it has been shown that injections of NPY directly into the carotid body circulation of cats increase activity of carotid sinus nerve afferents due to local vasoconstriction with consequent stagnant asphyxia [27]. However, no evidence for an increase in reflex sensitivity was found in the present experiments. Possibly, the injections of 5 /zg NPY directly into the isolated carotid sinus region of the cat [27] produced greater local concentrations of NPY in the carotid body than those caused systemically by injections of NPY used here.

The second reflex examined in this study was the arterial baroreceptor reflex. The relationship between SABP and PI is close to linear over that portion of its full extent examined here. The slope reflects the sensitivity of the baroreceptor- cardiodepressor reflex [34]. It was this relationship between SABP and PI which was used here to determine the arterial baroreceptor reflex sensitivity changes with time.

The first method used for evoking increases in

P1 in response to arterial baroreceptor stimulation was inflation of a balloon-tipped catheter positioned in the abdominal aorta. It was found after a bolus intravenous injection of NPY that the responses of PI to an increase in SABP were significantly reduced. This depression of reflex sensitivity, as indicated by significant decreases in the slope of the PI-SABP relations, was maintained for periods up to 50 rain after NPY injection. In experiments on unanaesthetised rabbits, Minson et al. [20] recently failed to find any inhibition of the baroreceptor reflex response, in contrast to our results. However, they did not test for an inhibitory action of NPY on the cardiac vagus by direct electrical stimulation, in order to confirm that this is similar in the rabbit to that in the dog. It has been observed in our laboratory that intravenous injections of NPY into anaesthetised rabbits have little or no inhibitory effect on the action of the electrically-stimulated vagus nerve on the heart (unpublished observations). It seems that NPY cannot be assumed to be an effective inhibitor of cardiac vagal action in all species. In the anaesthetised cat, for example, (where cardiac sympathetic nerves contain both NPY and galanin together with norardrenaline), NPY has little or no effect on cardiac vagal action whereas another sympathetic peptide, galanin, is strongly inhibitory [28].

In four animals the NPY-mediated inhibition of the steady-state arterial baroreceptor reflex, as evoked by inflation of the aortic balloon, was compared with that of vagal action at the heart. The alternative ' r amp ' method of stimulating the arterial baroreceptors, using intravenous injections of phenylephrine, was preferred for most tests here, however, because it provided a more rapid assessment of reflex behaviour. This method resembles closely that used in the original experiments performed by Smyth et al. [34], and is acknowledged to concentrate principally on the initial phase of the reflex response. However, as this initial phase depends largely on vagal mechanisms, its use here was appropriate. Comparison of the baroreceptor reflex response and the response to electrical stimulation of the vagus showed no significant difference in the magnitude of the evoked inhibition by NPY. However, the

time-course of the inhibition of the reflex response was significantly shorter than that of the vagal response. No significant difference in the NPY-mediated inhibition, in both MPI and T50, could be found between the reflex and vagal responses after propranolol. Thus, the major action of NPY in the studies presented here would appear to be an inhibition of reflexly evoked cardiac vagal activity. In experiments where microinjections of NPY have been delivered to the central nervous system of the rat, depressor and bradycardia or pressor and tachycardia responses have been evoked, depending on the site of injection [7,19,39]. Microinjections of NPY directly into the nucleus tractus solitarius, the site of the first synapse for the baroreceptor and chemoreceptor reflex pathways, produce a depressor response with bradycardia [4,38]. However, no evidence for this central action of NPY was found in this series of experiments. It is probable that such a large molecule as NPY (molecular weight 4272) does not cross the blood brain barrier after sys- temic injection.

The results reported here suggest that the NPY-mediated inhibition of prolongations of PI reflexly evoked by arterial baroreceptor stimulation, like those evoked by chemoreceptor stimulation, occur primarily at the vagal nerve endings to the heart, although additional sites of action cannot be ruled out. In experiments using a method similar to that described here, it has been shown that reflex responses in PI to baroreceptor stimulation are attenuated during exercise, mental stress and in hypertensive subjects [9,23,31,33]. Recent studies have shown that NPY is released in response to exercise and traumatic situations such as surgery [17,18,21]. Possibly, the decreased sensitivity of the baroreceptor reflex during stress could be, at least in part, attributable to release of endogenous NPY. The role of NPY in hypertension is unclear, yet there would appear to be increased levels of the peptide in arterial plasma and the adrenal medulla of stroke-prone spontaneously hypertensive rats [12,30]. While some of the decrease in sensitivity of the baroreceptor reflex when arterial pressure is held at an ele- vated level may be attributed to a decrease in the sensitivity of the baroreceptor afferents [24,32], a

177

possible inhibitory role of endogenous NPY cannot be discounted.

In conclusion, we have found that NPY in- hibits increases in PI evoked by stimulation of either the arterial chemoreceptors or baroreceptors and that this attenuation of the reflex responses can be adequately explained by the well- described inhibitory effect of NPY on cardiac vagal action at the nerve terminals in the heart.

Acknowledgements

This work was supported by the National Health and Medical Research Council of Aus- tralia and the National Heart Foundation of Aus- tralia. We thank Deborah McKay and Jane Bur- sill for expert technical assistance.

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