69
J. Phy8iol. (1979), 294, pp. 69-80 With 7 text-figure8 Printed in Great Britain
INHIBITION BY ANGIOTENSIN II OF BARORECEPTOR-EVOKED ACTIVITY IN CARDIAC VAGAL EFFERENT NERVES IN THE DOG
BY EUGENIE R. LUMBERS, D. I. McCLOSKEY AND ERICA K. POTTER From the School of Physiology and Pharmacology, University of New South Wales, Kensington, Sydney, Australia
(Received 17 October 1978) SUMMARY
1. Action potentials were recorded in single baroreceptor fibres dissected from the carotid sinus nerves in dogs during increases in blood pressure caused by i.v. injection of angiotensin II, and by i.v. injection of phenylephrine or inflation of an aortic balloon. Action potentials were recorded in single cardiac efferent fibres dissected from the right cervical vagus nerve in other dogs during increases in blood pressure caused by angiotensin II, and by phenylephrine or by inflation of an aortic balloon. 2. There was no difference in the discharge frequency of single carotid sinus baroreceptor fibres at any blood pressure when phenylephrine, balloon inflation, or angiotensin II were used to raise the pressure. 3. Activity in single cardiac vagal efferent fibres was increased when blood pressure was increased by phenylephrine or by inflation of an aortic balloon. However, when blood pressure rose by a comparable amount in response to angiotensin II, vagal firing decreased (three fibres), was little changed from control levels (four fibres), or increased less than it did in response to phenylephrine (one fibre). 4. It is concluded that while angiotensin II has no effect on baroreceptor sensitivity, it does inhibit vagal discharge which is evoked by stimulation of arterial baroreceptors. INTRODUCTION
Angiotensin II raises blood pressure by acting on structures within the central nervous system as well through its direct action on peripheral arterioles. Bickerton & Buckley (1961) did experiments on cross-perfused dogs and attributed the central pressor effect to stimulation of central sympathetic pathways. Since then it has been repeatedly shown in many species that injection of angiotensin II into a vertebral artery causes a greater increase in blood pressure than a similar dose given intravenously (see Severs & Daniels-Severs, 1973, for review). Scroop & Lowe (1969), in experiments on greyhounds, showed that vagal withdrawal as well as sympathetic activation may contribute to the central pressor effects of angiotensin II. Ismay, Lumbers & Stevens (1979) studied the effect of angiotensin II on the baroreceptor-cardiodepressor reflex in conscious sheep using
70
E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER the method of Smythe, Sleight & Pickering (1969). This method is particularly useful for demonstrating vagal effects. They used various automic blocking agents to show that administration of angiotensin II attenuates the usual vagally-mediated bradycardia which occurs in response to an increase in blood pressure. This attentuation appears to be an action of angiotensin II within the central nervous system. A peripheral action of angiotensin II on the baroreceptor nerve endings, which might have accounted for these findings, seemed unlikely as Sweet & Brody (1970) had failed to demonstrate an effect of intracarotid infusion of angiotensin II on the baroreceptor reflex when they used doses which did affect the reflex when given into a vertebral artery: also, topical application of angiotensin II to the carotid sinus had been claimed to have no effect on the arterial baroreceptors (McCubbin, Page & Bumpus, 1957). There were two parts to the present study. First, we looked for an effect of angiotensin II on the arterial baroreceptors by recording directly from carotid sinus baroreceptor fibres. Second, we recorded directly from cardiac vagal efferent fibres to see the effect of angiotensin II on vagal efferent activity. Part of this work has been reported in brief (Potter, Lumbers & McCloskey, 1978) METHODS
Experiments were performed on mongrel dogs of both sexes weighing from 7 to 14 kg. The animals were anaesthetized with i.v. chloralose (a-chloralose, British Drug Houses: 80 mg/kg) after induction with thiopentone. (Two of the animals described here were premedicated with morphine sulphate, 1 mg/kg, and then anaesthetized with chloralose without using thiopentone). In each dog the trachea was cannulated low in the neck and a nylon cannula was inserted into the lingual artery to measure blood pressure. A similar cannula was inserted into the femoral vein for the administration of anaesthetics and drugs. A balloon-tipped catheter was inserted through a femoral artery and advanced so that the inflatable balloon lay in the upper abdominal aorta. Rectal temperature was kept between 37 and 39 'C. The experimental animals breathed spontaneously in all experiments. Arterial pressure was recorded from the lingual artery using a Statham P23 AC transducer. Records of electrocardiogram and heart rate (beat by beat, triggered from the e.c.g.) were obtained using a Grass 7P4D preamplifier. A record of tracheal air flow was obtained by passing a wide (2 mm i.d.) nylon tube into the trachea and connecting it to a Grass 5PT5A volumetric pressure transducer. Blood pressure, heart rate, and tracheal air flow were recorded on a Grass polygraph. The entire pharynx and larynx were removed from just above the sternum up to the level of the hyoid bone, and the skin flaps were raised to make a pool which was then filled with liquid paraffin. For recording cardiac vagal efferent nerve activity, the vagus nerve on the right-hand side was cut, desheathed, and the central end was laid across a rigid, earthed, stainless-steel plate with a blackened upper surface. The nerve was then divided into filaments under a microscope with fine forceps. Neural activity in the filaments was recorded by lifting them, one by one, on to fine stainless-steel electrodes which were connected to preamplifiers (Neurolog NL 103/ NL 106: band pass between 10 Hz and 1 kHz), and then to a speaker and a storage oscilloscope. Filaments were dissected until single efferent units were recorded. Nerves were considered to be cardiac efferents if they demonstrated a cardiac rhythm, and an inhibition of their activity during inspiration (Jewett, 1964). Confirmation of this characterization was always obtained in one of two ways: (i) the nerve responded with a latency of 40-100 msec to single shocks applied to the central end of a carotid sinus nerve (Iriuchijima & Kumada, 1964), or (ii) the nerve increased its discharge while maintaining its cardiac and respiratory rhythms, in response to a 'mechanical' elevation of blood pressure produced by inflating the aortic balloon. Records of cardiac vagal efferent activity were obtained by direct photography from the oscilloscope screen. Alternatively, the cardiac efferent spikes were used to trigger a spike trigger (Neurolog NL 200).
ANGIOTENSIN AND VAGAL DISCHARGE
71
The trigger pulses were then counted in steps (usually 3-10 spikes/step), and every step of the counter raised the analogue output of a pulse integrator (Neurolog NL 600) by one unit. The integrator could be reset to zero at required intervals. The analogue output of the integrator was then recorded on the fourth channel of the Grass polygraph. By this method it was possible to obtain a record of spike frequency on a pen recorder. In experiments in which carotid sinus baroreceptor activity was recorded, the carotid sinus nerve was first identified by stimulating its central end (at 10 V, 50 Hz, 0-2 msec for 1-5 see) and recording the reflex reduction evoked in heart rate and blood pressure. The carotid sinus nerve was then cut close to its junction with the glossopharyngeal nerve, and desheathed. The procedure for isolating and recording single baroreceptor units was thereafter similar to that outlined above for single cardiac vagal efferents. Baroreceptor fibres were identified by their cardiac rhythm, with increasing activity when the blood pressure was raised 'mechanically', and decreased activity when the common carotid artery was occluded below the test carotid sinus. Angiotensin amide (Hypertensin, Ciba) was used in this study and the effects of its pressor actions on baroreceptor and cardiac vagal efferent activity were compared with the effects on these discharges of increases in blood pressure caused by inflation of an aortic balloon, or by injection of phenylephrine hydrochloride (Sterling Pharmaceuticals), a potent a-adrenergic vasoconstrictor with no direct effect on heart rate (Varma, Johnsen, Sherman & Youmans, 1960). Angiotensin (2-25 jog) and phenylephrine (20-100 fg) were given by i.v. injections: the doses used were sufficient to raise arterial blood pressure by 15-85 mmHg.
RESULTS
(i) Responses of baroreceptor fibres. Six single baroreceptor fibres dissected from the right carotid sinus nerves of three dogs were studied in detail. In all of these fibres there was no demonstrable difference in discharge at comparable blood pressure Phenylephrine B.P
150
(mmHg) Baroreceptor
-III
Aw,
~ip
spikes
~ ~
m
b
i
Angiotensin II B.P.
(mmHg) Baroreceptor
spikes
150IS 50 |1 1 a 11
MmI *
ih
.-~i~ W I sec ~~~~~~~~~~~1
Fig. 1. Dog, anaesthetized with chloralose, vagi cut: records of carotid sinus pressure and activity of a single carotid sinus baroreceptor fibre are shown. In the top panel, activity of the fibre is shown at three levels of blood pressure following injections of phenylephrine. It can be seen that as blood pressure increased (left to right), activity of the baroreceptor fibre increased. The bottom panel shows the activity of the same fibre when angiotensin II was used as the pressor agent. There was no demonstrable difference in the activity of this baroreceptor fibre at any blood pressure when angiotensin II rather than phenylephrine was used to increase blood pressure.
72
E. R. LUMBERS, D. 1. McCLOSKEY AND E. K. POTTER levels whether angiotensin II or phenylephrine was used as the pressor agent. A comparison of the responses of one of these baroreceptor fibres to increases in blood pressure caused by angiotensin II and by phenylephrine is given in Fig. 1, where individual baroreceptor spikes are shown. It can be seen that the discharge increased to a comparable degree as the blood pressure was increased by either agent. In the animal shown in Fig. 1 both vagi had been cut to minimize changes in heart rate in response to the change in blood pressure. 60
o Phenylephrine * Angiotensin 11
000
0000
50
50
0
~ ~ ~~~~~~~~~0 40
0
u! 40
so
0 0
04K0
0 00 0~so
0 0
0
* 0)O 0 0 30 -_0 -
00
0
000
0~~~~~~~ U)~~~~ 00 0o 0000
10 _
60
80
100 120 140 160 180 Systolic B.P. (mmHg) Fig. 2. Dog, anaesthetized with chloralose, vagi cut: discharge frequency of a single baroreceptor fibre is plotted against systolic pressure measured in the carotid sinus at various blood pressure levels. Open circles show the numbers of action potentials per second when phenylephrine was used to raise blood pressure: filled circles show the numbers of action potentials per second for the same fibre when angiotensin II was used as the pressor agent. There was no demonstrable difference in the activity of this fibre at any blood pressure level whether phenylephrine or angiotensin II was used to increase blood pressure.
A more detailed analysis of the results described above is shown for another fibre in Fig. 2. Again the vagi were cut. Systolic blood pressure was varied from 60 to 180 mmHg using a series of i.v. injections of both angiotensin II and phenylephrine: (pressures below 100 mmHg in the experiments shown in Fig. 1 and Fig. 2 were obtained by occluding the common carotid artery at the peak of a pressor response caused by either agent). Several responses were counted at each blood pressure level with each pressor agent: over a wide range of blood pressures no difference in the relation between baroreceptor frequency and blood pressure was found. This was so for all of the fibres tested and for the several multifibre preparations which were also tested in the course of the fibre splitting process. Vagotomy cannot be relied upon to abolish all heart rate responses to increases in
73 ANGIOTENSIN AND VAGAL DISCHARGE blood pressure: a degree of sympathetic withdrawal can also be expected (e.g. Bronk, 1933; Davis, McCloskey & Potter, 1977). The heart rate changes accompanying the pressor responses to angiotensin II and phenylephrine after vagotomy are different: this can be seen in Fig. 3. It is clear from Fig. 3, however, that the baroreceptor discharge is not influenced by the differing changes in heart rate but only by the level of arterial pressure (see also Arndt, Morgenstern & Samodelov, 1977). The differences in heart rate response which occur in vagotomized dogs (as well as H.R./min [5_ 90 300 Baroreceptor
spikes/5 sec
Ni 200
B.P.
(mmHg)
A
5
lOA 1 jigAlgAII
50
A
50 LgPhe
After propranolol
H.R./min
[
Baroreceptor
F
(mmHg)
50 F
spikes/5 sec B.P.
50 LA
1 min 50 ug Phe 10 pg A1 Fig. 3. Dog, heavily anaesthetized with chloralose, both vagi cut: records of heart rate (H.R.) (beat by beat, triggered from e.c.g.), baroreceptor discharge and carotid sinus blood pressure are shown during i.V. injections of angiotensin II (AII) and phenylephrine (Phe). Records made before administration of the fl-adrenergic blocking agent, propranolol, are in the upper panel and after propranolol in the lower panel. The increase in heart rate caused by intravenous injection of angiotensin (upper panel) is abolished by propranolol. Baroreceptor spikes were counted through 5 sec intervals: it can be seen that there was no difference in baroreceptor discharge rate at any blood pressure when either phenylephrine or angiotensin II was used as the pressor agent, eitherbefore fl-blockade (upper panel) or after fl-blockadel with propranolol (lower panel).
the sinus arrhythmia seen in the control state: e.g. upper panel, Fig. 3) can be attributed to fi-adrenergic mechanisms, as they are abolished by ,8-adrenergic blockade with propranolol (1 mg/kg: Fig. 3, lower panel). Note that Fig. 3 demonstrates ,8-adrenergic activation by angiotensin compared to ft sympathetic withdrawal evoked by a pressor dose of phenylephrine. (ii) Responses of cardiac vagal efferent fibres. Eight single cardiac vagal efferent fibres dissected from the right vagus nerve in eight dogs were studied in detail. In all fibres from all dogs cardiac vagal efferent activity increased as blood pressure
E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER rose in response to inflation of an aortic balloon or to administration of phenylephrine. This is the baroreceptor-cardiodepressor reflex. When blood pressure was raised by a comparable amount in response to administration of angiotensin II, vagal discharge was never as high as with phenylephrine or balloon inflation (P < 0.01, Student's t test, paired, two-tailed). Fig. 4 shows records of vagal action potentials in two of the fibres studied. Results from all eight fibres are summarized 74
Fibre 1
B
200 } 50
A II
Control a
Phe _
_
LEr
II.h V/agal a.p.
A
_
Fibre 2-
Control B.P.
A II
Phe
1200 1 00
Vagalya.p. 2 sec Fig. 4. Action potentials (a.p.) in two single cardiac vagal efferent fibres dissected from the right cervical vagi of two dogs are shown. Records of carotid sinus pressure and tracheal air-flow (inspiratory flow deflexion upwards) are also shown. The left panel for both fibres shows resting cardiac vagal efferent activity. The middle panel for both fibres shows the effect of a pressor dose of angiotensin II (A II) on cardiac vagal efferent activity. In the right-hand panel for both fibres blood pressure was increased by a comparable amount using phenylephrine (Phe). Cardiac vagal activity was markedly increased when blood pressure rose in response to phenylephrine, but not when it was raised by angiotensin fI. (Note the inspiratory pause in vagal firing shown for fibre 1: records from fibre 2 were taken during pauses between inspirations. Note also the marked cardiac rhythm shown for fibre 2 following phenylephrine injection.)
in Fig. 5, where it can be seen that during pressor responses caused by angiotensin II vagal discharge was actually reduced from control levels in three fibres, and was virtually unaffected in four others. For only one fibre did angiotensin II cause a pronounced increase in vagal discharge when blood pressure rose, and in that fibre the vagal firing increased by only about half as much as it did in response to a smaller rise in arterial pressure caused by phenylephrine. It is known that angiotensin II causes respiratory stimulation at any level of blood pressure when compared with phenylephrine or balloon inflation (Potter & McCloskey, 1979). It is also known that vagal discharge is inhibited during the inspiratory phase of breathing (Gandevia, McCloskey & Potter, 1978; and see upper panel of Fig. 4). Thus, the respiratory stimulant effects of angiotensin II will cause over-all vagal withdrawal by more frequently and completely interrupting cardiac
ANGIOTENSIN AND VAGAL DISCHARGE 75 vagal discharges. Nevertheless, the effects we report here are additional to those which are secondary to respiratory stimulation. The results plotted in Fig. 5 are levels of vagal discharge recorded in only the expiratory phases of breathing, so the vagal withdrawal they demonstrate cannot be attributed to more frequent interruption of vagal firing as a result of respiratory stimulation. This point is also evident on inspection of Fig. 4, where it is clear that vagal discharge between inspirations is inhibited by angiotensin II. The reported effects cannot be considered 400
-
C
. 300
-
U
X, O' 200
/ -
E)
10
0:
Phenylephrine Aortic balloon
*
Angiotensin 11
0
20
30 40 50 160 70 180 '90 Increase in B.P. (mmHg) Fig. 5. Vagal discharge (expressed as a percentage of control, preinjection rate) is plotted against change in blood pressure for all eight single cardiac vagal efferent fibres, studied in eight dogs. Lines join the responses for each individual fibre when blood pressure was increased to comparable levels by angiotensin II (filled circles), and either phenylephrine (open circles) or inflation of an aortic balloon (open squares). Rises in blood pressure caused by injection of phenylephrine or inflation of an aortic balloon caused an increase in vagal discharge in every fibre. Rises in blood pressure caused by injection of angiotensin II were associated with a fall in vagal discharge (three fibres), little change in vagal discharge (four fibres), or a rise in vagal discharge (one fibre): in the one fibre in which angiotensin II markedly increased vagal discharge, the vagal activity was less than that which occurred in response to injection of phenylephrine.
secondary to respiratory changes through other mechanisms. The same effects occur when angiotensin II is given after cutting both vagi, so that respiratory stimulation of intrathoracic receptors connected to the central nervous system through the vagus cannot be held responsible. Moreover, in several animals the pressor responses to both angiotensin II and phenylephrine were so prompt that there was no breath taken in either case before the peak of the pressor response (- 5 sec). In these animals vagal withdrawal following angiotensin II was still evident at this time. In these circumstances, differences in blood gas tensions following angiotensin II and phenylephrine would not be expected and so are unlikely to explain the inhibitory effects of angiotensin II. In three of the eight vagal fibres examined there was an initial, brief (3-5 sec)
76 E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER increase in discharge as blood pressure rose in response to angiotensin II, and this increase was comparable to that seen in response to phenylephrine or balloon inflation. In all three fibres, however, the increased level of firing on angiotensin was not maintained as it was with phenylephrine or balloon inflation, and by the time the peak of the pressor response had occurred (5-15 see), vagal firing was 750
Phe
E
A II
750
E 500
-d
i00
o
X250
X250
110 130 150 170 Systolic pressure (mmHg)
30 25
110 130 150 170 Systolic pressure (mmHg) Phe
05
-
A
II
-25-
~20 -20d.~~~~~~~~~~~~C c 215M
30
15
-
/5col
110 130 150 170 110 130 150 170 Systolic pressure (mmHg) Systolic pressure (mmHg) Fig. 6. Dog, anaesthetized with chloralose, left vagus intact. The upper two panels show regression lines for pulse interval plotted against systolic blood pressure, for points obtained during the rise of blood pressure to a peak following injection of phenylephrine (Phe, upper left) and angiotensin II (A II, upper right). The bottom two panels show regression lines for cardiac vagal efferent activity (a.p.) in impulses per second plotted against systolic blood pressure for the same periods following phenylephrine (lower left) and angiotensin II (lower right) see text.
clearly reduced below its initial peak and below the levels attained with phenylephrine or balloon inflation. The responses observed in these three fibres correlated well with changes in heart rate which occurred simultaneously and which were presumably mediated by the intact left vagus. Smythe et al. (1969) in man, and Ismay et al. (1979) in the sheep, reported similar poorly maintained bradyeardia in response to I.v. doses of angiotensin II. As might be expected, we were able to observe parallel changes in vagal discharge recorded from the right vagus and in cardiac pulse interval influenced by the intact left vagus in all these manoeuvres. Fig. 6 illustrates results obtained in an experiment in which it was possible to hold a cardiac vagal fibre long enough to make observa-
ANGIOTENSIN AND VAGAL DISCHARGE 77 tions on vagal firing and pulse interval during repeated administrations of phenylephrine and angiotensin II. During each pressor response systolic pressure, pulse interval and vagal discharge frequency were recorded beat by beat up to the peak of the blood pressure rise (counts during the inspiratory phases of breathing were omitted, see above). Linear regressions were then calculated for the relations between pulse interval and systolic pressure, and between vagal discharge and systolic pressure: the resulting regression lines are shown in Fig. 6. The limits of the lines shown are the pre-injection, and peak, blood pressures. This form of analysis is widely used in the analysis of baroreceptor reflex behaviour (e.g. Bristow, Brown, Cunningham, Goode, Howson & Sleight, 1971). As the 'sensitivity' of the baroreceptor-cardiodepressor reflex is given by the slopes of such regression lines when arterial pressure is raised by a simple peripheral vasoconstrictor, baroreflex 'sensitivity' could be calculated and assessed statistically here (see also, Discussion). The slope of each line was taken as a number and the means and standard deviations of these numbers were calculated for each condition, and then compared by Student's t test (unpaired, two-tailed). For the data shown in Fig. 6, the slopes of pulse intervalblood pressure lines, and the vagal discharge-blood pressure lines obtained with phenylephrine were significantly greater (P < 0*001) than those obtained with angiotensin II. Other evidence of vagal inhibition by angiotensin. In the experiments described here vagal discharge was recorded from the cut central end of the right vagus while the left vagus remained intact. The left vagus has, because of its anatomical distribution, a more powerful effect than the right vagus on the atrio-ventricular node, and a less powerful effect on the sino-atrial node (Garey, 1911). Therefore, the atrio-ventricular block which occurred repeatedly in three of our dogs and occasionally in others during pressor responses to phenylephrine can be attributed to baroreceptor-mediated vagal bombardment of the atrio-ventricular node. At no time did we observe atrioventricular block following administration of angiotensin II, even in the most susceptible animals. This further supports the conclusion that angiotensin inhibits vagal firing. DISCUSSION
The present study provides direct evidence that angiotensin II inhibits cardiac vagal efferent activity which is evoked by stimulation of arterial baroreceptors. The effects we report are not due to an action of angiotensin II on baroreceptor sensory endings. This was to be expected following the failure of Sweet & Brody (1970) to produce cardiovascular effects with intracarotid injections of angiotensin II in doses which evoked pressor responses when given into a vertebral artery, and the failure of McCubbin et al. (1957) to evoke systemic cardiovascular responses by applying angiotensin directly into the wall of the carotid sinus. The direct recordings of baroreceptor activity reported here confirm this expectation. Scroop & Lowe (1969) originally proposed a vagal inhibitory action for angiotensin II on the basis of pharmacological experiments in anaesthetized greyhounds. Ismay et al. (1979) studied conscious sheep and showed that the bradycardia evoked when blood pressure was raised by angiotensin II was less marked than when the same increase in blood pressure was produced by phenylephrine. Again a
78 E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER vagal inhibitory action of angiotensin was proposed, and again pharmacological evidence was given to support the proposal. Ismay et al. (1979) also pointed out that phenylephrine and angiotensin II had been previously noted to give differing heart rate responses in man (Smythe et al. 1969), although a vagal inhibitory action of antiotensin II had not been specifically suggested to account for this. Of course, vagal discharge must first be present before its inhibition can be demonstrated: loss of vagal tone in the experimental animal, especially under anaesthesia, may therefore A
Phe
a:
0
7'
C.~~A
Blood pressure 7. Discussion Fig. Figure - see text.
account for the previous failure of many investigators to have noted the vagal inhibitory action of angiotensin (see Severs &z Daniels-Severs, 1973, for review). The slope of the relation between cardiac pulse interval (or vagal firing rate) and arterial pressure is widely used as an indicator of the sensitivity of the baroreceptorcardiodepressor reflex (e.g. Bristow et al. 1971; Korner, Shaw, West, Oliver &r Hilder, 1973). When the relation is determined, beat by beat, as blood pressure rises in response to an injection of phenylephrine, the vagal component of the reflex is especially conspicuous (Korner, 1971). Thus the effects seen when this form of analysis is used to examine the actions of angiotensin II show well the inhibitory action of angiotensin II (Ismay et al. 1979; Fig. 6). Nevertheless, the reduction in slope seen when angiotensin II was used to raise blood pressure may not indicate a reduction of the sensitivity of the baroreceptor reflex. Angiotensin II might conceivably act on central baroreflex pathways in such a way as to shift the operating relation between blood pressure and vagal discharge (the so-called 'set-point': see Korner, 1971; Streatfeild, Davidson &; McCloskey, 1977) without altering baroreflex: sensitivity. A bolus dose of angiotensin II given intravenously might, therefore, reach central baroreflex pathways in gradually increasing concentration, thereby shifting
ANGIOTENSIN AND VAGAL DISCHARGE 79 the so-called 'set-point' of the reflex with concentration while not necessarily altering its sensitivity. Such a possibility is depicted in Fig. 7. Line A represents baroreflex behaviour as it would be revealed on administration of phenylephrine: the parallel (dotted) lines to the right of line A indicate the effects of one conceivable action of angiotensin II, a gradual shift of 'set point' with time, without any alteration in baroreflex sensitivity. If angiotensin II were to have such an action, the relations between pulse interval (or vagal discharge) and blood pressure following its administration in a bolus dose might be described by lines B or C, that is, by a series of points obtained on successively shifted, but parallel, lines. The point of the argument pursued with reference to Fig. 7 is that angiotensin II may inhibit vagal activity without necessarily reducing the sensitivity of the baroreceptor-cardiodepressor reflex. Because angiotensin II has centrally mediated as well as peripheral cardiovascular effects, its effects on baroreceptor sensitivity can be assessed only when it is present in steady concentration. Therefore, the effects of angiotensin II on baroreflex sensitivity remain to be determined. The vagal inhibitory action of angiotensin II is probably exerted within the central nervous system on structures supplied via the vertebral arteries in, or near to, the area postrema. Considerable evidence now exists that such structures are involved in other centrally mediated cardiovascular effects (Severs & DanielsSevers, 1973) and the work of Scroop & Lowe (1969) on intravertebral infusions of angiotensin II suggest that the vagal inhibitory effects are exerted in the same region. The doses of angiotensin II used in the present study were chosen to produce rises in blood pressure which are sufficient, when achieved by other means, to increase cardiac vagal discharge. Such doses appear large in comparison with the physiological range of blood levels of angiotensin II (8-56 pg/ml.: Boyd, Landon & Peart, 1967; Lumbers & Reid, 1977). However, there is considerable dilution of an i.v. bolus dose, and about 50 % of such a dose is removed in a single circulation (Hodge, Ng & Vane, 1967). Furthermore, peak blood levels of a bolus dose are achieved only transiently, and may not be well reflected at a receptor site separated from the blood by a diffusion barrier. These matters must all be studied further before the physiological significance of the findings reported here can be fully assessed. This work was supported by a grant from the National Heart Foundation of Australia. Miss Diane Madden provided expert technical assistance.
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E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER
DAVIS, A. L., MCCLOSKEY, D. I. & POTrER, E. K. (1977). Respiratory modulation of-baroreceptor and chemoreceptor reflexes affecting heart rate through the sympathetic nervous system. J. Phyaiol. 272, 691-703. GANDEVIA, S. C., MCCLOSKEY, D. I. & POTTER, E. K. (1978). Inhibition of baroreceptor and chemoreceptor reflexes on heart rate by afferents from the lungs. J. Phy8iol. 276, 369-381. GAREY, W. E. (1911). Studies on the extrinsic and intrinsic nerve mechanisms of the heart. Am. J. Phyaiol. 28, 330. HODGE, R. L., NG, K. K. F. & VANE, J. R. (1967). Disappearance of angiotensin from the circulation of the dog. Nature, Lond. 215, 138-144. IRIUcHIJiA, J. & KUMADA, M. (1964). Activity of single vagal fibres efferent to the heart. Jap. J. Phy8iol. 14, 479-487. IsMAY, M. J. A., LuMBERS, E. R. & STEVENS, A. D. (1979). The action of angiotensin fl on the baroreflex response of the conscious ewe and the conscious foetus. J. Physiol. 288, 467-481. JEWETr, D. L. (1964). Activity of single efferent fibres in the cervical vagus nerve of the dog, with special reference to possible cardio-inhibitory fibres. J. Physiol. 175, 321-357. KORNER, P. I. (1971). Integrative neural cardiovascular control. Physiol. Rev. 51, 312-367. KORNER, P. I., LHAW, J., WEST, M. J., OLIVER, J. R. & HILDER, R. G. (1973). Integrative reflex control of heart rate in the rabbit during hypoxia and hyperventilation. Circulation Res. 33, 63-73. LumBERS, E. R. & REID, G. C. (1977). Effects of vaginal delivery and Caesarian section on plasma renin activity and angiotensin II levels in human umbilical cord blood. Biologia Neonat. 31, 127-134. McCUBBIN, J. W., PAGE, I. H. & BuMPus, F. M. (1957). Effect of synthetic angiotensin on the carotid sinus circulation. Circulation Res. 5, 458-460. POTTER, E. K., LUMBERS, E. R. & MCCLOSKEY, D. I. (1978). Inhibition of cardiac vagal efferent activity by intravenous administration of angiotensin II. Proceedings of the Australian Society for Clinical and Experimental Pharmacologists, December, Abstr. 69. Melbourne: ASCEP. POTTER, E. K. & MCCLOSKEY, D. I. (1979). Respiratory stimulation by angiotensin II. Reep. Physiol. 36, 367-373. ScRoop, G. C. & LowE, R. D. (1969). Efferent pathways of the cardiovascular response to vertebral artery infusions of angiotensin in the dog. Clin. Sci. 37, 605-619. SEVERS, W. B. & DANIELS-SEVERS, A. E. (1973). Effects of angiotensin on the central nervous system. Pharmac. Rev. 25, 41-449. SMYTHE, H. S., SLEIGHT, P. & PICKERING, G. W. (1969). A quantitative method of assessing baroreflex sensitivity. Circulation Res. 24, 109-121. STREATFEILD, K. A., DAVIDSON, N. S. & MCCLOSKEY, D. I. (1977). Muscular reflex and baroreflex influences on heart rate during isometric contractions. Cardiovasc. Res. 11, 87-93. SWEET, C. S. & BRODY, M. J. (1970). Central inhibition of reflex vasodilatation by angiotensin and reduced renal pressure. Am. J. Physiol. 219, 1751-1758. VARMA, S., JOHNSEN, S. D., SHERMAN, D. E. & YOUMANS, W. B. (1960). Mechanisms of inhibition of heart rate by phenylephrine. Circulation Res. 8, 1182-1186.
J. Phy8iol. (1979), 294, pp. 69-80 With 7 text-figure8 Printed in Great Britain
INHIBITION BY ANGIOTENSIN II OF BARORECEPTOR-EVOKED ACTIVITY IN CARDIAC VAGAL EFFERENT NERVES IN THE DOG
BY EUGENIE R. LUMBERS, D. I. McCLOSKEY AND ERICA K. POTTER From the School of Physiology and Pharmacology, University of New South Wales, Kensington, Sydney, Australia
(Received 17 October 1978) SUMMARY
1. Action potentials were recorded in single baroreceptor fibres dissected from the carotid sinus nerves in dogs during increases in blood pressure caused by i.v. injection of angiotensin II, and by i.v. injection of phenylephrine or inflation of an aortic balloon. Action potentials were recorded in single cardiac efferent fibres dissected from the right cervical vagus nerve in other dogs during increases in blood pressure caused by angiotensin II, and by phenylephrine or by inflation of an aortic balloon. 2. There was no difference in the discharge frequency of single carotid sinus baroreceptor fibres at any blood pressure when phenylephrine, balloon inflation, or angiotensin II were used to raise the pressure. 3. Activity in single cardiac vagal efferent fibres was increased when blood pressure was increased by phenylephrine or by inflation of an aortic balloon. However, when blood pressure rose by a comparable amount in response to angiotensin II, vagal firing decreased (three fibres), was little changed from control levels (four fibres), or increased less than it did in response to phenylephrine (one fibre). 4. It is concluded that while angiotensin II has no effect on baroreceptor sensitivity, it does inhibit vagal discharge which is evoked by stimulation of arterial baroreceptors. INTRODUCTION
Angiotensin II raises blood pressure by acting on structures within the central nervous system as well through its direct action on peripheral arterioles. Bickerton & Buckley (1961) did experiments on cross-perfused dogs and attributed the central pressor effect to stimulation of central sympathetic pathways. Since then it has been repeatedly shown in many species that injection of angiotensin II into a vertebral artery causes a greater increase in blood pressure than a similar dose given intravenously (see Severs & Daniels-Severs, 1973, for review). Scroop & Lowe (1969), in experiments on greyhounds, showed that vagal withdrawal as well as sympathetic activation may contribute to the central pressor effects of angiotensin II. Ismay, Lumbers & Stevens (1979) studied the effect of angiotensin II on the baroreceptor-cardiodepressor reflex in conscious sheep using
70
E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER the method of Smythe, Sleight & Pickering (1969). This method is particularly useful for demonstrating vagal effects. They used various automic blocking agents to show that administration of angiotensin II attenuates the usual vagally-mediated bradycardia which occurs in response to an increase in blood pressure. This attentuation appears to be an action of angiotensin II within the central nervous system. A peripheral action of angiotensin II on the baroreceptor nerve endings, which might have accounted for these findings, seemed unlikely as Sweet & Brody (1970) had failed to demonstrate an effect of intracarotid infusion of angiotensin II on the baroreceptor reflex when they used doses which did affect the reflex when given into a vertebral artery: also, topical application of angiotensin II to the carotid sinus had been claimed to have no effect on the arterial baroreceptors (McCubbin, Page & Bumpus, 1957). There were two parts to the present study. First, we looked for an effect of angiotensin II on the arterial baroreceptors by recording directly from carotid sinus baroreceptor fibres. Second, we recorded directly from cardiac vagal efferent fibres to see the effect of angiotensin II on vagal efferent activity. Part of this work has been reported in brief (Potter, Lumbers & McCloskey, 1978) METHODS
Experiments were performed on mongrel dogs of both sexes weighing from 7 to 14 kg. The animals were anaesthetized with i.v. chloralose (a-chloralose, British Drug Houses: 80 mg/kg) after induction with thiopentone. (Two of the animals described here were premedicated with morphine sulphate, 1 mg/kg, and then anaesthetized with chloralose without using thiopentone). In each dog the trachea was cannulated low in the neck and a nylon cannula was inserted into the lingual artery to measure blood pressure. A similar cannula was inserted into the femoral vein for the administration of anaesthetics and drugs. A balloon-tipped catheter was inserted through a femoral artery and advanced so that the inflatable balloon lay in the upper abdominal aorta. Rectal temperature was kept between 37 and 39 'C. The experimental animals breathed spontaneously in all experiments. Arterial pressure was recorded from the lingual artery using a Statham P23 AC transducer. Records of electrocardiogram and heart rate (beat by beat, triggered from the e.c.g.) were obtained using a Grass 7P4D preamplifier. A record of tracheal air flow was obtained by passing a wide (2 mm i.d.) nylon tube into the trachea and connecting it to a Grass 5PT5A volumetric pressure transducer. Blood pressure, heart rate, and tracheal air flow were recorded on a Grass polygraph. The entire pharynx and larynx were removed from just above the sternum up to the level of the hyoid bone, and the skin flaps were raised to make a pool which was then filled with liquid paraffin. For recording cardiac vagal efferent nerve activity, the vagus nerve on the right-hand side was cut, desheathed, and the central end was laid across a rigid, earthed, stainless-steel plate with a blackened upper surface. The nerve was then divided into filaments under a microscope with fine forceps. Neural activity in the filaments was recorded by lifting them, one by one, on to fine stainless-steel electrodes which were connected to preamplifiers (Neurolog NL 103/ NL 106: band pass between 10 Hz and 1 kHz), and then to a speaker and a storage oscilloscope. Filaments were dissected until single efferent units were recorded. Nerves were considered to be cardiac efferents if they demonstrated a cardiac rhythm, and an inhibition of their activity during inspiration (Jewett, 1964). Confirmation of this characterization was always obtained in one of two ways: (i) the nerve responded with a latency of 40-100 msec to single shocks applied to the central end of a carotid sinus nerve (Iriuchijima & Kumada, 1964), or (ii) the nerve increased its discharge while maintaining its cardiac and respiratory rhythms, in response to a 'mechanical' elevation of blood pressure produced by inflating the aortic balloon. Records of cardiac vagal efferent activity were obtained by direct photography from the oscilloscope screen. Alternatively, the cardiac efferent spikes were used to trigger a spike trigger (Neurolog NL 200).
ANGIOTENSIN AND VAGAL DISCHARGE
71
The trigger pulses were then counted in steps (usually 3-10 spikes/step), and every step of the counter raised the analogue output of a pulse integrator (Neurolog NL 600) by one unit. The integrator could be reset to zero at required intervals. The analogue output of the integrator was then recorded on the fourth channel of the Grass polygraph. By this method it was possible to obtain a record of spike frequency on a pen recorder. In experiments in which carotid sinus baroreceptor activity was recorded, the carotid sinus nerve was first identified by stimulating its central end (at 10 V, 50 Hz, 0-2 msec for 1-5 see) and recording the reflex reduction evoked in heart rate and blood pressure. The carotid sinus nerve was then cut close to its junction with the glossopharyngeal nerve, and desheathed. The procedure for isolating and recording single baroreceptor units was thereafter similar to that outlined above for single cardiac vagal efferents. Baroreceptor fibres were identified by their cardiac rhythm, with increasing activity when the blood pressure was raised 'mechanically', and decreased activity when the common carotid artery was occluded below the test carotid sinus. Angiotensin amide (Hypertensin, Ciba) was used in this study and the effects of its pressor actions on baroreceptor and cardiac vagal efferent activity were compared with the effects on these discharges of increases in blood pressure caused by inflation of an aortic balloon, or by injection of phenylephrine hydrochloride (Sterling Pharmaceuticals), a potent a-adrenergic vasoconstrictor with no direct effect on heart rate (Varma, Johnsen, Sherman & Youmans, 1960). Angiotensin (2-25 jog) and phenylephrine (20-100 fg) were given by i.v. injections: the doses used were sufficient to raise arterial blood pressure by 15-85 mmHg.
RESULTS
(i) Responses of baroreceptor fibres. Six single baroreceptor fibres dissected from the right carotid sinus nerves of three dogs were studied in detail. In all of these fibres there was no demonstrable difference in discharge at comparable blood pressure Phenylephrine B.P
150
(mmHg) Baroreceptor
-III
Aw,
~ip
spikes
~ ~
m
b
i
Angiotensin II B.P.
(mmHg) Baroreceptor
spikes
150IS 50 |1 1 a 11
MmI *
ih
.-~i~ W I sec ~~~~~~~~~~~1
Fig. 1. Dog, anaesthetized with chloralose, vagi cut: records of carotid sinus pressure and activity of a single carotid sinus baroreceptor fibre are shown. In the top panel, activity of the fibre is shown at three levels of blood pressure following injections of phenylephrine. It can be seen that as blood pressure increased (left to right), activity of the baroreceptor fibre increased. The bottom panel shows the activity of the same fibre when angiotensin II was used as the pressor agent. There was no demonstrable difference in the activity of this baroreceptor fibre at any blood pressure when angiotensin II rather than phenylephrine was used to increase blood pressure.
72
E. R. LUMBERS, D. 1. McCLOSKEY AND E. K. POTTER levels whether angiotensin II or phenylephrine was used as the pressor agent. A comparison of the responses of one of these baroreceptor fibres to increases in blood pressure caused by angiotensin II and by phenylephrine is given in Fig. 1, where individual baroreceptor spikes are shown. It can be seen that the discharge increased to a comparable degree as the blood pressure was increased by either agent. In the animal shown in Fig. 1 both vagi had been cut to minimize changes in heart rate in response to the change in blood pressure. 60
o Phenylephrine * Angiotensin 11
000
0000
50
50
0
~ ~ ~~~~~~~~~0 40
0
u! 40
so
0 0
04K0
0 00 0~so
0 0
0
* 0)O 0 0 30 -_0 -
00
0
000
0~~~~~~~ U)~~~~ 00 0o 0000
10 _
60
80
100 120 140 160 180 Systolic B.P. (mmHg) Fig. 2. Dog, anaesthetized with chloralose, vagi cut: discharge frequency of a single baroreceptor fibre is plotted against systolic pressure measured in the carotid sinus at various blood pressure levels. Open circles show the numbers of action potentials per second when phenylephrine was used to raise blood pressure: filled circles show the numbers of action potentials per second for the same fibre when angiotensin II was used as the pressor agent. There was no demonstrable difference in the activity of this fibre at any blood pressure level whether phenylephrine or angiotensin II was used to increase blood pressure.
A more detailed analysis of the results described above is shown for another fibre in Fig. 2. Again the vagi were cut. Systolic blood pressure was varied from 60 to 180 mmHg using a series of i.v. injections of both angiotensin II and phenylephrine: (pressures below 100 mmHg in the experiments shown in Fig. 1 and Fig. 2 were obtained by occluding the common carotid artery at the peak of a pressor response caused by either agent). Several responses were counted at each blood pressure level with each pressor agent: over a wide range of blood pressures no difference in the relation between baroreceptor frequency and blood pressure was found. This was so for all of the fibres tested and for the several multifibre preparations which were also tested in the course of the fibre splitting process. Vagotomy cannot be relied upon to abolish all heart rate responses to increases in
73 ANGIOTENSIN AND VAGAL DISCHARGE blood pressure: a degree of sympathetic withdrawal can also be expected (e.g. Bronk, 1933; Davis, McCloskey & Potter, 1977). The heart rate changes accompanying the pressor responses to angiotensin II and phenylephrine after vagotomy are different: this can be seen in Fig. 3. It is clear from Fig. 3, however, that the baroreceptor discharge is not influenced by the differing changes in heart rate but only by the level of arterial pressure (see also Arndt, Morgenstern & Samodelov, 1977). The differences in heart rate response which occur in vagotomized dogs (as well as H.R./min [5_ 90 300 Baroreceptor
spikes/5 sec
Ni 200
B.P.
(mmHg)
A
5
lOA 1 jigAlgAII
50
A
50 LgPhe
After propranolol
H.R./min
[
Baroreceptor
F
(mmHg)
50 F
spikes/5 sec B.P.
50 LA
1 min 50 ug Phe 10 pg A1 Fig. 3. Dog, heavily anaesthetized with chloralose, both vagi cut: records of heart rate (H.R.) (beat by beat, triggered from e.c.g.), baroreceptor discharge and carotid sinus blood pressure are shown during i.V. injections of angiotensin II (AII) and phenylephrine (Phe). Records made before administration of the fl-adrenergic blocking agent, propranolol, are in the upper panel and after propranolol in the lower panel. The increase in heart rate caused by intravenous injection of angiotensin (upper panel) is abolished by propranolol. Baroreceptor spikes were counted through 5 sec intervals: it can be seen that there was no difference in baroreceptor discharge rate at any blood pressure when either phenylephrine or angiotensin II was used as the pressor agent, eitherbefore fl-blockade (upper panel) or after fl-blockadel with propranolol (lower panel).
the sinus arrhythmia seen in the control state: e.g. upper panel, Fig. 3) can be attributed to fi-adrenergic mechanisms, as they are abolished by ,8-adrenergic blockade with propranolol (1 mg/kg: Fig. 3, lower panel). Note that Fig. 3 demonstrates ,8-adrenergic activation by angiotensin compared to ft sympathetic withdrawal evoked by a pressor dose of phenylephrine. (ii) Responses of cardiac vagal efferent fibres. Eight single cardiac vagal efferent fibres dissected from the right vagus nerve in eight dogs were studied in detail. In all fibres from all dogs cardiac vagal efferent activity increased as blood pressure
E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER rose in response to inflation of an aortic balloon or to administration of phenylephrine. This is the baroreceptor-cardiodepressor reflex. When blood pressure was raised by a comparable amount in response to administration of angiotensin II, vagal discharge was never as high as with phenylephrine or balloon inflation (P < 0.01, Student's t test, paired, two-tailed). Fig. 4 shows records of vagal action potentials in two of the fibres studied. Results from all eight fibres are summarized 74
Fibre 1
B
200 } 50
A II
Control a
Phe _
_
LEr
II.h V/agal a.p.
A
_
Fibre 2-
Control B.P.
A II
Phe
1200 1 00
Vagalya.p. 2 sec Fig. 4. Action potentials (a.p.) in two single cardiac vagal efferent fibres dissected from the right cervical vagi of two dogs are shown. Records of carotid sinus pressure and tracheal air-flow (inspiratory flow deflexion upwards) are also shown. The left panel for both fibres shows resting cardiac vagal efferent activity. The middle panel for both fibres shows the effect of a pressor dose of angiotensin II (A II) on cardiac vagal efferent activity. In the right-hand panel for both fibres blood pressure was increased by a comparable amount using phenylephrine (Phe). Cardiac vagal activity was markedly increased when blood pressure rose in response to phenylephrine, but not when it was raised by angiotensin fI. (Note the inspiratory pause in vagal firing shown for fibre 1: records from fibre 2 were taken during pauses between inspirations. Note also the marked cardiac rhythm shown for fibre 2 following phenylephrine injection.)
in Fig. 5, where it can be seen that during pressor responses caused by angiotensin II vagal discharge was actually reduced from control levels in three fibres, and was virtually unaffected in four others. For only one fibre did angiotensin II cause a pronounced increase in vagal discharge when blood pressure rose, and in that fibre the vagal firing increased by only about half as much as it did in response to a smaller rise in arterial pressure caused by phenylephrine. It is known that angiotensin II causes respiratory stimulation at any level of blood pressure when compared with phenylephrine or balloon inflation (Potter & McCloskey, 1979). It is also known that vagal discharge is inhibited during the inspiratory phase of breathing (Gandevia, McCloskey & Potter, 1978; and see upper panel of Fig. 4). Thus, the respiratory stimulant effects of angiotensin II will cause over-all vagal withdrawal by more frequently and completely interrupting cardiac
ANGIOTENSIN AND VAGAL DISCHARGE 75 vagal discharges. Nevertheless, the effects we report here are additional to those which are secondary to respiratory stimulation. The results plotted in Fig. 5 are levels of vagal discharge recorded in only the expiratory phases of breathing, so the vagal withdrawal they demonstrate cannot be attributed to more frequent interruption of vagal firing as a result of respiratory stimulation. This point is also evident on inspection of Fig. 4, where it is clear that vagal discharge between inspirations is inhibited by angiotensin II. The reported effects cannot be considered 400
-
C
. 300
-
U
X, O' 200
/ -
E)
10
0:
Phenylephrine Aortic balloon
*
Angiotensin 11
0
20
30 40 50 160 70 180 '90 Increase in B.P. (mmHg) Fig. 5. Vagal discharge (expressed as a percentage of control, preinjection rate) is plotted against change in blood pressure for all eight single cardiac vagal efferent fibres, studied in eight dogs. Lines join the responses for each individual fibre when blood pressure was increased to comparable levels by angiotensin II (filled circles), and either phenylephrine (open circles) or inflation of an aortic balloon (open squares). Rises in blood pressure caused by injection of phenylephrine or inflation of an aortic balloon caused an increase in vagal discharge in every fibre. Rises in blood pressure caused by injection of angiotensin II were associated with a fall in vagal discharge (three fibres), little change in vagal discharge (four fibres), or a rise in vagal discharge (one fibre): in the one fibre in which angiotensin II markedly increased vagal discharge, the vagal activity was less than that which occurred in response to injection of phenylephrine.
secondary to respiratory changes through other mechanisms. The same effects occur when angiotensin II is given after cutting both vagi, so that respiratory stimulation of intrathoracic receptors connected to the central nervous system through the vagus cannot be held responsible. Moreover, in several animals the pressor responses to both angiotensin II and phenylephrine were so prompt that there was no breath taken in either case before the peak of the pressor response (- 5 sec). In these animals vagal withdrawal following angiotensin II was still evident at this time. In these circumstances, differences in blood gas tensions following angiotensin II and phenylephrine would not be expected and so are unlikely to explain the inhibitory effects of angiotensin II. In three of the eight vagal fibres examined there was an initial, brief (3-5 sec)
76 E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER increase in discharge as blood pressure rose in response to angiotensin II, and this increase was comparable to that seen in response to phenylephrine or balloon inflation. In all three fibres, however, the increased level of firing on angiotensin was not maintained as it was with phenylephrine or balloon inflation, and by the time the peak of the pressor response had occurred (5-15 see), vagal firing was 750
Phe
E
A II
750
E 500
-d
i00
o
X250
X250
110 130 150 170 Systolic pressure (mmHg)
30 25
110 130 150 170 Systolic pressure (mmHg) Phe
05
-
A
II
-25-
~20 -20d.~~~~~~~~~~~~C c 215M
30
15
-
/5col
110 130 150 170 110 130 150 170 Systolic pressure (mmHg) Systolic pressure (mmHg) Fig. 6. Dog, anaesthetized with chloralose, left vagus intact. The upper two panels show regression lines for pulse interval plotted against systolic blood pressure, for points obtained during the rise of blood pressure to a peak following injection of phenylephrine (Phe, upper left) and angiotensin II (A II, upper right). The bottom two panels show regression lines for cardiac vagal efferent activity (a.p.) in impulses per second plotted against systolic blood pressure for the same periods following phenylephrine (lower left) and angiotensin II (lower right) see text.
clearly reduced below its initial peak and below the levels attained with phenylephrine or balloon inflation. The responses observed in these three fibres correlated well with changes in heart rate which occurred simultaneously and which were presumably mediated by the intact left vagus. Smythe et al. (1969) in man, and Ismay et al. (1979) in the sheep, reported similar poorly maintained bradyeardia in response to I.v. doses of angiotensin II. As might be expected, we were able to observe parallel changes in vagal discharge recorded from the right vagus and in cardiac pulse interval influenced by the intact left vagus in all these manoeuvres. Fig. 6 illustrates results obtained in an experiment in which it was possible to hold a cardiac vagal fibre long enough to make observa-
ANGIOTENSIN AND VAGAL DISCHARGE 77 tions on vagal firing and pulse interval during repeated administrations of phenylephrine and angiotensin II. During each pressor response systolic pressure, pulse interval and vagal discharge frequency were recorded beat by beat up to the peak of the blood pressure rise (counts during the inspiratory phases of breathing were omitted, see above). Linear regressions were then calculated for the relations between pulse interval and systolic pressure, and between vagal discharge and systolic pressure: the resulting regression lines are shown in Fig. 6. The limits of the lines shown are the pre-injection, and peak, blood pressures. This form of analysis is widely used in the analysis of baroreceptor reflex behaviour (e.g. Bristow, Brown, Cunningham, Goode, Howson & Sleight, 1971). As the 'sensitivity' of the baroreceptor-cardiodepressor reflex is given by the slopes of such regression lines when arterial pressure is raised by a simple peripheral vasoconstrictor, baroreflex 'sensitivity' could be calculated and assessed statistically here (see also, Discussion). The slope of each line was taken as a number and the means and standard deviations of these numbers were calculated for each condition, and then compared by Student's t test (unpaired, two-tailed). For the data shown in Fig. 6, the slopes of pulse intervalblood pressure lines, and the vagal discharge-blood pressure lines obtained with phenylephrine were significantly greater (P < 0*001) than those obtained with angiotensin II. Other evidence of vagal inhibition by angiotensin. In the experiments described here vagal discharge was recorded from the cut central end of the right vagus while the left vagus remained intact. The left vagus has, because of its anatomical distribution, a more powerful effect than the right vagus on the atrio-ventricular node, and a less powerful effect on the sino-atrial node (Garey, 1911). Therefore, the atrio-ventricular block which occurred repeatedly in three of our dogs and occasionally in others during pressor responses to phenylephrine can be attributed to baroreceptor-mediated vagal bombardment of the atrio-ventricular node. At no time did we observe atrioventricular block following administration of angiotensin II, even in the most susceptible animals. This further supports the conclusion that angiotensin inhibits vagal firing. DISCUSSION
The present study provides direct evidence that angiotensin II inhibits cardiac vagal efferent activity which is evoked by stimulation of arterial baroreceptors. The effects we report are not due to an action of angiotensin II on baroreceptor sensory endings. This was to be expected following the failure of Sweet & Brody (1970) to produce cardiovascular effects with intracarotid injections of angiotensin II in doses which evoked pressor responses when given into a vertebral artery, and the failure of McCubbin et al. (1957) to evoke systemic cardiovascular responses by applying angiotensin directly into the wall of the carotid sinus. The direct recordings of baroreceptor activity reported here confirm this expectation. Scroop & Lowe (1969) originally proposed a vagal inhibitory action for angiotensin II on the basis of pharmacological experiments in anaesthetized greyhounds. Ismay et al. (1979) studied conscious sheep and showed that the bradycardia evoked when blood pressure was raised by angiotensin II was less marked than when the same increase in blood pressure was produced by phenylephrine. Again a
78 E. R. LUMBERS, D. I. McCLOSKEY AND E. K. POTTER vagal inhibitory action of angiotensin was proposed, and again pharmacological evidence was given to support the proposal. Ismay et al. (1979) also pointed out that phenylephrine and angiotensin II had been previously noted to give differing heart rate responses in man (Smythe et al. 1969), although a vagal inhibitory action of antiotensin II had not been specifically suggested to account for this. Of course, vagal discharge must first be present before its inhibition can be demonstrated: loss of vagal tone in the experimental animal, especially under anaesthesia, may therefore A
Phe
a:
0
7'
C.~~A
Blood pressure 7. Discussion Fig. Figure - see text.
account for the previous failure of many investigators to have noted the vagal inhibitory action of angiotensin (see Severs &z Daniels-Severs, 1973, for review). The slope of the relation between cardiac pulse interval (or vagal firing rate) and arterial pressure is widely used as an indicator of the sensitivity of the baroreceptorcardiodepressor reflex (e.g. Bristow et al. 1971; Korner, Shaw, West, Oliver &r Hilder, 1973). When the relation is determined, beat by beat, as blood pressure rises in response to an injection of phenylephrine, the vagal component of the reflex is especially conspicuous (Korner, 1971). Thus the effects seen when this form of analysis is used to examine the actions of angiotensin II show well the inhibitory action of angiotensin II (Ismay et al. 1979; Fig. 6). Nevertheless, the reduction in slope seen when angiotensin II was used to raise blood pressure may not indicate a reduction of the sensitivity of the baroreceptor reflex. Angiotensin II might conceivably act on central baroreflex pathways in such a way as to shift the operating relation between blood pressure and vagal discharge (the so-called 'set-point': see Korner, 1971; Streatfeild, Davidson &; McCloskey, 1977) without altering baroreflex: sensitivity. A bolus dose of angiotensin II given intravenously might, therefore, reach central baroreflex pathways in gradually increasing concentration, thereby shifting
ANGIOTENSIN AND VAGAL DISCHARGE 79 the so-called 'set-point' of the reflex with concentration while not necessarily altering its sensitivity. Such a possibility is depicted in Fig. 7. Line A represents baroreflex behaviour as it would be revealed on administration of phenylephrine: the parallel (dotted) lines to the right of line A indicate the effects of one conceivable action of angiotensin II, a gradual shift of 'set point' with time, without any alteration in baroreflex sensitivity. If angiotensin II were to have such an action, the relations between pulse interval (or vagal discharge) and blood pressure following its administration in a bolus dose might be described by lines B or C, that is, by a series of points obtained on successively shifted, but parallel, lines. The point of the argument pursued with reference to Fig. 7 is that angiotensin II may inhibit vagal activity without necessarily reducing the sensitivity of the baroreceptor-cardiodepressor reflex. Because angiotensin II has centrally mediated as well as peripheral cardiovascular effects, its effects on baroreceptor sensitivity can be assessed only when it is present in steady concentration. Therefore, the effects of angiotensin II on baroreflex sensitivity remain to be determined. The vagal inhibitory action of angiotensin II is probably exerted within the central nervous system on structures supplied via the vertebral arteries in, or near to, the area postrema. Considerable evidence now exists that such structures are involved in other centrally mediated cardiovascular effects (Severs & DanielsSevers, 1973) and the work of Scroop & Lowe (1969) on intravertebral infusions of angiotensin II suggest that the vagal inhibitory effects are exerted in the same region. The doses of angiotensin II used in the present study were chosen to produce rises in blood pressure which are sufficient, when achieved by other means, to increase cardiac vagal discharge. Such doses appear large in comparison with the physiological range of blood levels of angiotensin II (8-56 pg/ml.: Boyd, Landon & Peart, 1967; Lumbers & Reid, 1977). However, there is considerable dilution of an i.v. bolus dose, and about 50 % of such a dose is removed in a single circulation (Hodge, Ng & Vane, 1967). Furthermore, peak blood levels of a bolus dose are achieved only transiently, and may not be well reflected at a receptor site separated from the blood by a diffusion barrier. These matters must all be studied further before the physiological significance of the findings reported here can be fully assessed. This work was supported by a grant from the National Heart Foundation of Australia. Miss Diane Madden provided expert technical assistance.
REFERENCES ARNDT, J. O., MORGENSTERN, J. & SAMODELOV, L. (1977). The physiologically relevant information regarding systemic blood pressure encoded in the carotid sinus baroreceptor discharge pattern. J. Physiol. 268, 775-791. BICKERTON, R. K. & BUCKLEY, J. P. (1961). Evidence for a central mechanism in angiotensin induced hypertension. Proc. Soc. exp. Biol. Med. 106, 834-836. BOYD, G. W., LANDON, J. & PEART, W. S. (1967). Radioimmunoassay for determining plasma levels of angiotensin II in man. Lancet iR, 1002-1005. BRISTOW, J. D., BROWN, E. B., CUNNINGHAM, D. J. C., GOODE, R. C., HOWSON, M. B. & SLEIGHT, P. (1971). The effects of hypercapnia, hypoxia and ventilation on the baroreflex regulation of pulse interval. J. Physiol. 216, 281-302. BRONK, D. W. (1933). The nervous mechanism of cardiovascular control. Harvey Lect., 245-262.
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