pharmacological separation of cardio-accelerator and vagal inhibitory capacities of sympathetic...

Journal of the Autonomic Nervous System, 43 (1993) 7-16 7 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00

JANS 01371

Pharmacological separation of cardio-accelerator and vagal inhibitory capacities of sympathetic nerves

Mark Moriarty, Erica K. Potter and D.I. McCloskey Prince of Wales Medical Research Institute, Prince of Wales Hospital, Randwick, Sydney, NSW, Australia

(Received 30 June 1992) (Revision received 10 September 1992)

(Accepted 7 October 1992)

Key words: Chlor i sondamine ; N e u r o p e p t i d e Y; Rese rp ine ; Sympathet ic ; Vagus

Abstract

Prolonged attenuation of vagal action at the heart, proposed to be due to release of the sympathetic cotransmitter neuropeptide Y (NPY), follows stimulation of cardiac sympathetic nerves. It has been shown that pretreatment with reserpine depletes cardiac and neuronal stores of both noradrenaline and NPY, while combined pretreatment with reserpine and the ganglion blocking agent chlorisondamine reduces depletion of NPY, while still depleting noradrenaline. The effects of reserpine pretreatment and combined chlorisondamine and reserpine pretreatment on the inhibition of cardiac vagal action evoked by cardiac sympathetic nerve stimulation (16 Hz, 2 min) were compared in anaesthetised dogs. In dogs with no pretreatment (n = 6), sympathetic stimulation evoked an immediate cardio-acceleration, and a prolonged inhibition of cardiac vagal action, with a maximum percent inhibition (MPI) and time to half-recovery (Ts0) of 78 -1- 6% and 16 + 2 min respectively. In dogs pretreated with reserpine (n = 6, 1 mg/kg, 24 h), the immediate cardio-acceleration (ANOVA, P < 0.01), and the magnitude (MPI = 31.8%, ANOVA, P < 0.001) and duration (Ts0 = 6 + 1 min, ANOVA, P < 0.05) of inhibition of cardiac vagal action following sympathetic stimulation were significantly attenuated. In dogs with combined chlorisondamine (n = 5, 2 mg/kg, 48 and 24 h) and reserpine pretreatment, there was again significantly reduced cardio-acceleration (ANOVA, P < 0.01), but the inhibition of cardiac vagal action following sympathetic stimulation did not significantly differ from untreated animals (MPI = 79 + 8%, Ts0 = 21 + 6 min). Intravenous injections of NPY (25-50/~g/kg) evoked prolonged inhibition of cardiac vagal action in untreated and both groups of pretreated animals. These experiments indicate that the cardio-accelerator and vagal inhibitory capacities of sympathetic nerve stimulation can be separated, and are consistent with the sympathetic vagal inhibitory factor being NPY.

Introduction

Exper imen t s on anaes the t i sed dogs have shown

that fol lowing a pe r iod of cardiac sympathet ic

Correspondence to: E.K. Potter, Prince of Wales Medical Research Institute, Prince of Wales Hospital, Randwick, 2031, Sydney, NSW, Australia.

nerve s t imulat ion the re is an a t t enua t ion of vagal

act ion at the hear t [20-23,27,28]. This inhibi t ion

of vagal act ion is resis tant to a - and /3-adrenoc-

ep tor blockade, and is not mimicked by bolus

in t ravenous inject ions of no rad rena l ine [20-

23,27]. However , bolus in t ravenous inject ions of

N P Y do r ep roduce this p ro longed inhibi t ion of

cardiac vagal action. The action is a presynapt ic

one, on pos tgangl ionic vagal nerve fibres [21].

Neuropeptide Y (NPY) is a 36 amino acid peptide originally isolated from extracts of porcine brain [25,26]. The pattern of distribution of NPY suggests that this peptide may play an important role in cardiovascular regulation, as NPY is colo- calised with noradrenaline in sympathetic nerve fibres to the heart and blood vessels [1,2,4,7]. There is evidence from both the pig and the dog that direct stimulation of the cardiac sympathetic nerves leads to an increase in the overflow of NPY-like immunoreactive material into the coronary sinus [24,29]. It is this NPY-like substance released after cardiac sympathetic nerve stimulation which is believed to produce the prolonged inhibition of vagal action.

Recently it has been shown that reserpine pretreatment depletes cardiac and neuronal stores not only of noradrenaline, but NPY also [9-12,16]. However, evidence suggests that reserpine-induced depletion of NPY differs in some respects from the depletion of noradrenaline. The reserpine-induced depletion of NPY is tissue-specific, with neuronal stores of NPY in heart and skeletal muscle extensively depleted [9,11,12,16]. Also, surgical decentralization of ganglia, or pharmacological ganglionic blockade, markedly reduces the reserpine-induced depletion of NPY, while still producing significant depletion of noradrenaline [10,12,13,18,19]. This is said to occur by reducing the neuronal depletion of NPY after reserpine by reducing the neural traffic in the sympathetic nerves [10].

On the basis of such pretreatment protocols in dogs, one could apply a test of the hypothesis that the vagal inhibitory transmitter is indeed NPY. Hence, in animals pretreated with reserpine it would be expected that with sympathetic stimulation, both vagal inhibition and cardio-accelerator and pressor responses, would be diminished. However, in animals which had combined chlorisondamine and reserpine pretreatment, it would be predicted that any reduction in vagal inhibition would be significantly less than that seen following reserpine alone, while reduction of the cardio-accelerator and pressor responses would be similar to those seen after reserpine pretreatment. The present study tested these predictions.

Materials and Methods

Surgical preparation Seventeen mongrel dogs of both sexes, weight

range 3-8 kg, were used. The animals were anaesthetised initially with intravenous thiopen- tone (15 mg/kg, Pentothal, Abbott Laboratories, Sydney, Australia), followed by intravenous a- chloralose (50-100 mg/kg, Sigma Chemical Co., St. Louis, MO, USA). The trachea was cannulated low in the neck, and connected to a positive pressure ventilator (Harvard). A femoral vein was cannulated for administration of drugs and sup- plements of chloralose. A femoral artery was cannulated for continuous blood pressure recording via a Gould Statham (P23AC) pressure trans- ducer connected to one channel of a Grass poly- graph. The electrocardiogram was recorded through subcutaneous needle electrodes and dis- played on a storage oscilloscope. The electrocardiogram was used to obtain beat-by-beat pulse interval (PI), after processing with Neurolog mod- ules (Digitimer, England). PI was preferred for indicating changes in heart rate as it has been shown that the relationship between PI and vagal stimulation frequency is a linear one [6,17]. Deep abdominal temperature was maintained in the range 37-38°C using heating pads.

Both the left and right vagus nerves were isolated and cut low in the neck. The right vagus nerve was laid across platinum electrodes for stimulation. Supramaximal stimuli ( ~ 40 V, 1 ms), from a Grass $88 stimulator with stimulus isola- tion unit, were delivered to the right vagus nerve in trains of 5 s duration, at intervals of 30 s. The frequency of vagal stimulation (2-3 Hz) was cho- sen to give a submaximal increase in PI, between 300 and 400 ms.

To expose the right cardiac sympathetic nerves, the right foreleg and scapula were reflected for- wards by tying and cutting the muscles attaching the right scapula to the thorax. The second rib was exposed and removed, and the right stellate ganglion identified and dissected free. The right ansa subclavia was laid across platinum electrodes for supramaximal stimulation ( ~ 20 V, 1 ms) by another channel of the stimulator. Stimu-

lation periods were for 2 minutes at a frequency of 16 Hz. The left sympathetic innervation was left intact, as this side has been shown to be less effective in evoking inhibition of vagally induced changes in PI [28].

Experimental protocol The experiments were conducted with the ap-

proval of the institutional animal care and ethics committee, and in accordance with the Code of Practice for Use of Animals of the National Health and Medical Research Council of Aus- tralia. The 17 animals were divided into 3 experimental groups. The first group (6 dogs) were the control, and had no pretreatment. The second group (6 dogs) were pretreated with reserpine (Sigma). One dose of reserpine (1 mg/kg), dissolved in 20% ascorbic acid, was injected in- traperitoneally 24 h prior to the experiment [18]. The third group (5 animals) were pretreated with both chlorisondamine (Ecolid, Ciba) and reserpine. Two injections of chlorisondamine (2 mg/kg), dissolved in 0.9% saline, were given in- traperitoneally, 48 and 24 h prior to the experiment [10]. One injection of reserpine (1 mg/kg) was given 24 h prior to the experiment. Nearly all animals pretreated with reserpine developed some diarrhoea, but were otherwise not distressed.

The aim of the study was to compare the vagal inhibitory effect of right cardiac sympathetic nerve stimulation and bolus intravenous injections of NPY (human, Pacific Biotechnology, Sydney) among the 3 groups of dogs. The control animals received NPY injections of 35 + 3 /xg/kg (range 25-44/zg/kg, 5.9-10.3 nmole/kg), the reserpine treated animals received 43 + 2 /xg/kg (range 40-50/zg/kg, 9.4-11.7 nmole/kg) and the combined chlorisondamine and reserpine treated animals received 39 + 3 /xg/kg (range 31-44/zg/kg, 7.3-10.3 nmole/kg). The variable doses resulted from giving a standard gravimetric dose to animals of different weight. There was no significant difference between the three doses on a dose/body weight basis (ANOVA).

To enable comparisons of the vagal inhibitory responses, the maximum percent inhibition (MPI) and the time to half-recovery (Ts0) for these responses were calculated. Control stimuli were

delivered to the right vagus to obtain repro- ducible increases in pulse interval (API). Then, after stimulation of the right ansa subclavia (2 min, 16 Hz), or an intravenous injection of NPY, intermittent stimulation of the right 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 [3]. The API 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 or the cessation of sympathetic stimulation. This time coincides with the most frequently observed time of maximum inhibition. The use of the regression equation to calculate the maximally inhibited zaPI value was preferred to simply recording the smallest zaPI observed, because the regression equation made the figure recorded dependent upon the full se- ries of points observed after intervention rather than one single observed point. MPI was calculated using the equation:

API¢ - API t MPI = × 100

APIc

where API c and API t are the control and treatment zaPI responses respectively. The Ts0 was also calculated from the regression line. In the pretreated animals, two and sometimes three, sympathetic stimulation periods were performed to test for any further depletion of neurotrans- mitters. No new intervention was performed until API had returned to control levels. The cardio- acceleration occurring at the time of sympathetic stimulation was also recorded (as a decrease in baseline PI). This effect was presumed to be due to noradrenaline release, as NPY has no chronotropic effects [20]. Changes in systolic arterial blood pressure were also recorded.

Analysis of variance (ANOVA) followed by Newman-Keuls tests were used to test significance of values between untreated and pretreated groups. A significance level of P < 5% was considered to be statistically significant. All

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Results

All the results for these experiments are sum- marised in Table I. In untreated animals, following an intervention-free period after surgery, resting PI was 490 + 40 ms and resting systolic arterial blood pressure was 135 + 6 mm Hg. A period of cardiac sympathetic nerve stimulation produced immediate tachycardia (see below), and a marked inhibition of cardiac vagal action in the 6 control animals. Following cardiac sympathetic nerve stimulation for 2 min, the PI responses to electrical stimulation of the vagus nerve were attenuated, with a MPI of 78 + 6%. This attenuation was a prolonged one, with a Ts0 of 16 + 2 min for API responses to return to control levels (Table I).

In the 6 animals which had been pretreated with reserpine alone, there was a significant increase in resting PI, to 620 + 20 ms (ANOVA,

11

P < 0.01) and a significant decrease in resting blood pressure to 101 + 7 mm Hg (ANOVA, P < 0.05). In the reserpine pretreated animals, a greatly reduced inhibition of vagal action was seen after cardiac sympathetic nerve stimulation in 5 of the 6 animals, and no inhibition in the remaining dog (see Fig. 1). The MPI for this attenuation of PI responses to electrical stimulation of the vagus nerve in all 6 dogs was 31 + 8%. This level of inhibition was significantly lower than that obtained for untreated animals (ANOVA, P < 0.001). The Ts0 for this response was 6 + 1 min. This was also significantly less than the Ts0 observed in untreated animals (ANOVA, P < 0.05). In all 6 animals pretreated with reserpine alone, a second period of cardiac sympathetic nerve stimulation was tested. The MPI of this subsequent stimulation period on cardiac vagal action was significantly less than the inhibition resulting from the first stimulation period (paired t-test, P < 0.001).

The third group of animals (n = 5) were pretreated with both chlorisondamine and reserpine. These animals had a significantly higher resting

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Fig. 1. Recordings of pulse interval (PI) and arterial blood pressure (BP) from an anaesthetised dog which had been pretreated only with reserpine (1 mg/kg) 24 h prior to the experiment. The top panel shows the effect of a 2 min period of cardiac sympathetic nerve stimulation (S16) on the increases in PI evoked by electrical stimulation of the right vagus nerve. For all 3 periods of sympathetic stimulation, no inhibition of cardiac vagal action was observed. Note also the small pressor and tachycardia responses during the stimulation period. The bottom panel shows that the prolonged attenuation of cardiac vagal action following a bolus

intravenous injection of NPY (44 p.g/kg) remains intact.

12

PI at 670 _+ 40 ms when compared with untreated animals (ANOVA, P < 0.01), although the resting blood pressure of 121 +_ 9 mm Hg was not significantly different from control. In these animals, cardiac sympathetic nerve stimulation elicited a prolonged attenuation of cardiac vagal action. The MPI and Ts0 for this response were 79 _+ 8% and 21 _+ 6 min respectively. There was no significant difference between these values and those obtained in unt rea ted animals (ANOVA). However, the value for MPI was significantly higher than that obtained for the animals pretreated with reserpine alone (ANOVA, P < 0.001). Also, the I"50 for these animals was significantly larger than that for reserpine treated animals (ANOVA, P < 0.05). Subsequent cardiac sympathetic nerve stimulations still evoked prolonged cardiac vagal attenuation, although these values were significantly lower than those ob-

tained for the first stimulation period (ANOVA, P < 0.05). Fig. 2 shows an example of one such experiment performed in a dog pretreated with chlorisondamine and reserpine.

At the time of cardiac sympathetic nerve stimulation in untreated animals there was a marked tachycardia and pressor response (Table I). This cardio-accelerator response at the time of sympathetic stimulation was significantly reduced in both reserpine and combined reserpine and chlorisondamine pretreated animals (ANOVA, P < 0.01). Also, the increase in arterial blood pressure was significantly reduced in reserpine and combined reserpine and chlorisondamine pretreated animals (ANOVA, P < 0.01).

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Fig. 2. Recordings of pulse interval (PI) and arterial blood pressure (BP) from an anaesthetised dog which had been pretreated with chlorisondamine (2 mg/kg) 48 and 24 h prior to the experiment, and with reserpine (1 mg/kg) 24 h prior to the experiment. The top 2 panels show the effects of 3 consecutive periods of cardiac sympathetic nerve stimulation on the increases in PI evoked by electrical stimulation of the vagus nerve. In each case, sympathetic stimulation produced a prolonged inhibition of cardiac vagal action, with a MPI of 70%, 50% and 56% respectively, and a "1"50 of 10, 11 and 9 min respectively. However, each stimulation period evoked only small pressor and tachycardia responses. The bottom panel shows the prolonged attenuation of cardiac vagal

action following a bolus intravenous injection of NPY (33/zg/kg).

13

TABLE II

Effects of reserpine and combined chlorisondamine and reserpine pretreatment on attenuation of cardiac vagal action evoked by intravenous NPY injections

NPY

MPI Ts0 ABP (%) (min) (mm Hg)

Control (n = 6) 6 0 + 6 3 4 + 5 205:4

Reserpine (n = 6) 4 6 + 4 325:6 335:5

Chlori- sondamine + reserpine (n = 5) 725:7 2 9 + 5 4 7 + 8 *

MPI, maximum percent inhibition; Ts0, half-time to recovery; ABP, increase in systolic arterial blood pressure. * P < 0.05, for significance of pretreated values compared with controls by analysis of variance. All values listed are mean + s tandard error of the mean.

the heart (Table II). There was no significant difference in the magnitude or timecourse of this inhibition between the untreated and pretreated groups (ANOVA). However, the increase in arterial blood pressure evoked by intravenous NPY was significantly larger in the combined reserpine and chlorisondamine pretreated animals when compared with untreated and reserpine pretreated animals (ANOVA, P < 0.05).

Discussion

The results reported here show that the immediate cardio-acceleration and the prolonged inhibition of cardiac vagal action caused by sympathetic stimulation can be functionally separated following appropriate pharmacological pretreatment. This result is consistent with the two responses being mediated by separate cotransmit- ters.

The inhibition of cardiac vagal action following a period of sympathetic stimulation has been attributed to NPY released from those sympathetic fibres [20-23,27,28]. Accompanying the cardiac sympathetic nerve stimulation are cardio-accelerator and pressor responses, pre-

sumably attributable to the actions of released noradrenaline. On the basis of this reasoning, the immediate cardio-acceleration and the pressor responses were used as a measure of noradrenaline release, and the prolonged inhibition of cardiac vagal action was used as a measure of NPY release. In dogs pretreated with reserpine, the tachycardia and pressor responses during sympathetic stimulation, and the attenuation of cardiac vagal action following it, were significantly reduced. This would suggest that both n0- radrenaline and NPY had been depleted from the cardiac sympathetic nerves. In animals which had been pretreated with both reserpine and the ganglionic blocking agent chlorisondamine, the tachycardia and pressor responses during sympathetic stimulation were significantly reduced, but the inhibition of cardiac vagal action following sympathetic stimulation was intact, suggesting that noradrenaline had been depleted while leaving NPY stores relatively intact. There was no significant difference between the levels of vagal inhibition obtained in those animals and in untreated ones.

It has recently been shown that pretreatment with reserpine depletes cardiac neuronal stores not only of noradrenaline but also of NPY. Im- munohistochemical studies on guinea pigs have shown that pretreatment with reserpine (5 mg/kg) 24 h prior to sacrifice reduced significantly the right atrial levels of NPY-like immunoreactivity and noradrenaline respectively by 42% and 94% [10] and 77% and 84% [16]. In another study on guinea pigs, using the same dosage 8 h prior to sacrifice, cardiac NPY-like immunoreactivity and noradrenaline content were reduced by 76% and 86% respectively [12]. Immunohistochemical studies have also revealed significant depletion of NPY immunoreactivity caused by reserpine in nerve fibres to the myocardium and related blood vessels, and to blood vessels in the lungs and skeletal muscle [9,11,12].

However, evidence suggests that there are dif- ferences in the way that reserpine acts to deplete the NPY and noradrenaline stores in nerve termi- nals. The reserpine induced depletion of NPY is tissue-specific, and extensive in heart and skeletal muscle [9,11,12,16]. The reserpine induced deple-

14

tion of NPY is slower in onset and, in particular, interruption of ganglionic transmission using the ganglion blocking agent chlorisondamine pre- vents depletion of NPY whilst allowing noradrenaline depletion to proceed [10,12]. It is proposed that ganglionic transmission must be intact for reserpine induced depletion of NPY to take place, and that once noradrenaline stores become depleted, and hence the negative feedback inhibition that this transmitter has on NPY release is removed [8,14], then NPY will be released to an extent that nerve terminal stores are gradually depleted by continuing sympathetic neural activity. Treatment with chlorisondamine alone does not significantly alter neuronal stores of noradrenaline or NPY in the myocardium [10,12].

This proposal has been supported by func- tional studies in the perfused cat spleen [13,15], perfused gracilis muscle preparations in the dog [18] and in renal preparations in pigs [19]. In each case reserpine pretreatment led to a significant decrease in the release of noradrenaline and NPY-like immunoreactivity upon sympathetic stimulation to that organ. However, in preparations which had been surgically decentralised be- fore reserpine administration, the release of NPY-like immunoreactivity upon nerve stimulation returned to control levels, and in the case of the cat spleen and pig kidney, this release ex- ceeded control levels. This potentiated release of NPY was attributed to the removal of the inhibitory effects of noradrenaline on NPY release via presynaptic a-adrenoceptors.

The absence of cardiac vagal inhibition following a period of sympathetic nerve stimulation in animals pretreated with reserpine cannot be attributed to a reduced effectiveness of released NPY. Bolus injections of NPY in reserpine pretreated animals and in untreated ones were simi- larly effective in attenuating cardiac vagal action. Also, a second period of sympathetic stimulation in animals pretreated with reserpine resulted in a significantly smaller inhibition of cardiac vagal action, with a MPI of only 11 _+ 7%, suggesting that NPY stores had further been depleted. In studies on untreated dogs, Hall et al. [5] showed that it took many separate sympathetic stimulation periods of 1 min each to deplete NPY and

reduce the MPI of cardiac vagal inhibition to levels comparable with those reported here for animals pretreated with reserpine.

In animals which had been pretreated here with both reserpine and chlorisondamine, the second period of sympathetic stimulation also resulted in a significantly smaller cardiac vagal inhibition (Table I). A result with possibly a similar underlying cause has been reported by Lund- berg et al. [15], who showed that in the isolated perfused spleen of cats pretreated with chlorisondamine and reserpine, splenic venous NPY-like immunoreactivity following splenic nerve stimulation (10 Hz, 2 min) was significantly reduced for a second stimulation period, and for each subsequent stimulation period. However, in the experiments reported here, a third sympathetic stimulation period resulted in a cardiac vagal inhibition with a MPI of 54 _+ 4%. This was not significantly different from the second sympathetic stimulation period, possibly because impairment of NPY stores in these animals was not as marked as in reserpine treated animals.

Chlorisondamine pretreatment did not impair the cardiac slowing effects of cervical (preganglionic) vagal nerve stimulation. Presumably, the ganglionic blockade had worn off in the 24 h between the most recent administration of this ganglionic blocker, and the experimental period (see also [12]). However, the results would indicate that the dosages of chlorisondamine used in these experiments were effective in significantly reducing the reserpine-induced depletion of NPY.

Intravenous injections of similar doses of NPY in both untreated and pretreated animals evoked cardiac vagal inhibition of similar magnitude and timecourse. However, the increase in arterial blood pressure observed in chlorisondamine and reserpine treated animals following NPY injection was significantly larger than that observed in control and reserpine treated animals. The mech- anism responsible for this is unclear.

In summary, dogs pretreated with reserpine showed significant reductions in the tachycardia and pressor responses to cardiac sympathetic nerve stimulation, and the attenuation of cardiac vagal action, suggesting depletion of both noradrenaline and NPY from the sympathetic nerves.

Animals which had been pretreated with the ganglion blocking drug chlorisondamine in addition to reserpine also showed reduced tachycardia and pressor responses, suggesting depletion of noradrenaline. In these, however, cardiac vagal inhibition was not significantly different from untreated animals. It has been shown that combined chlorisondamine and reserpine treatment leaves NPY stores relatively intact [10,12]. Thus, the results presented here are consistent with the proposal that NPY is a mediator of the sympathetic evoked vagal attenuation.

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 Beryl O'Mara for typing the manuscript, and Deborah McKay and Jane Bur- sill for expert technical assistance.

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15

8 Lundberg, J.M., ,~ngg~rd, A., Theodorsson-Norheim, E. and Pernow, J., Guanethidine-sensitive release of neuropeptide Y-like immunoreactivity in the cat spleen by sympathetic nerve stimulation, Neurosci. Lett., 52 (1984) 175-180.

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13 Lundberg, J.M., Fried, G., Pernow, T., Theodorsson- Norheim, E. and ,~ngg~rd, A., NPY - A mediator of reserpine-resistant, non-adrenergic vasoconstriction in cat spleen after preganglionic denervation? Acta Physiol. Scand., 126 (1986) 151-152.

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pharmacological separation of cardio-accelerator and vagal inhibitory capacities of sympathetic...

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