Effect of clonidine on responses to cardiac nerve stimulation as a function of impulse frequency and stimulus duration in vagotomized dogs

European Journal of Pharmacology 29 (1974) 182-186

© North-Holland Publishing Company

Short communication E F F E C T OF C L O N I D I N E ON RESPONSES TO C A R D I A C N E R V E S T I M U L A T I O N AS A F U N C T I O N O F I M P U L S E F R E Q U E N C Y A N D S T I M U L U S D U R A T I O N IN VAGOTOMIZED DOGS Ronald D. ROBSON and Michael J. ANTONACCIO Research Department, Pharmaceuticals Division, Ciba-Geigy Corporation, Summit, New Jersey 07901, U.S.A.

Received 19 August 1974, accepted 18 September 1974 R.D. ROBSON and M.J. ANTONACCIO,Effect of clonidine on responses to cardiac nerve stimulation as a function of impulse frequency and stimulus duration in vagotomized dogs, European J. Pharmacol. 29 (1974) 182-186. Using optimum voltageand. variable periods of right cardiac nerve stimulation in vagotomizeddogs, clonidine (30 /~g/kg i.v.) reliably inhibited the tachycardia to 0.3 and 1 Hz, but antagonized only responses to brief periods of stimulation at impulse frequencies of 3 and 10 Hz. Clonidine extended the stimulus duration necessary to cause maximum tachycardia, particularly at the lower frequencies, and did not significantly depress the magnitude of response except at 0.3 Hz. The effect of clonidine on stimulus duration, which was apparently due to a-adrenoceptor activation since it was prevented by phentolamine, was not obtained with guanethidine (0.9 mg/kg i.v.), which caused good inhibition of the maximum tachycardia attainable at all selected frequencies. Cardiac nerve function

Clonidine

Guanethidine

1. Introduction In an earlier study, we found that clonidine caused marked inhibition of positive chronotropic responses to cardiac nerve stimulation in dogs with functional baroreceptor reflexes, but failed to significantly inhibit responses to any impulse frequency of stimulation in vagotomized dogs (Antonaccio and Robson, 1973). Since clonidine enhances efferent vagal activity in response to baroreceptor activation (Robson and Kaplan, 1969; Robson et al., 1969; Nayler and Stone, 1970; Kobinger and Walland, 1971; 1972), and cardiac nerve stimulation elevates blood pressure, the apparent sympathetic nerve blockade after clonidine in non.vagotomized dogs probably resulted from concurrent vagal hyperactivity. While Scriabine and Stavorski (1973) confirmed the importance of a vagal component, their results in vagotomized dogs differed from ours since they reported that clonidine inhibited responses to low (1.58 and 5 Hz) but not high (15.8 Hz) impulse frequencies of cardiac nerve stimulation. In considering their criticism of our results in vago-

Phentolamine

Stimulus duration

tomized dogs, we feel that procedural differences, notably in the selection of stimulation parameters, rather than reinterpretation of data may better explain the discordant finding.

2. Materials and methods Mongrel dogs of either sex weighing between 8.0 and 15.7 kg were anesthetized with sodium pentobarbital (32.5 mg/kg i.v.) and prepared for cardiac nerve stimulation as previously described (Antonaccio and Robson, 1973). All dogs were bilaterally vagotomized. Curves relating heart rate to frequency of stimula. tion of the cardiac nerve were constructed from val ues obtained with square wave pulses of varying voltage as indicated in the text, 5-msec duration and applied at 0.3, 1, 3 and 10 Hz in a non-cumulative fashion. The duration and frequency was varied as noted in the text. Results are expressed as the mean value -+ S.E.M.

183

R.D. Robson, M.J. Antonaccio, Clonidine and cardiac nerve function

The significance of differences was assessed by Student's t-test. Doses of clonidine and phentolamine are expressed as the hydrochloride and guanethidine as the monosulfate salts.

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The post-ganglionic cardiac nerve was stimulated at several impulse frequencies for periods up to 32 sec (fig. 1) using a constant, maximal current strength of 3 V (see fig. 2). With a frequency of 0.3 Hz, nerve stimulation for periods beyond 8 sec caused little further increase in response and the minor tachycardia was prevented b y 30 #g/kg i.v. o f clonidine (fig. 1A). As impulse frequency was increased, stimu-

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Fig. 1. Tachycardia in response to variable periods of nerve stimulation at impulse frequencies of 0.3, 1, 3 and 10 Hz before (e . . . . . . e) and 30 min after 30 ~g/kg i.v. of clonidine (o o) (n = 5).

lus duration became a more important determinant of heart rate response. Responses at each duration o f I Hz stimulation were uniformly inhibited by clonidine (fig. 1B), but only responses to brief periods o f stimulation were inhibited when frequencies o f 3 and 10 Hz were applied (fig. IC and D, respectively). The influence of varying the voltage intensity was studied when stimulation was maintained for sufficient duration to permit m a x i m u m tachycardia at each impulse frequency. The optimum intensity was established as 3 V since the f r e q u e n c y - r e s p o n s e curve (fig. 2B) was superior to that obtained using

184

R.D. Robson, M.J. Antonaccio, Clonidine and cardiac nerve function

Table 1 Effect of various drug treatments on stimulus duration necessary to cause maximum tachycardia at various impulse frequencies of cardiac nerve stimulation in vagotomized, anesthetized dogs. Treatment

None

n

Time (sec) to maximum response at indicated frequencies

20

0.3 Hz

1.0 Hz

3.0 Hz

10.0 Hz

23.2 -+ 1.4

24.3 -+ 0.7

18.5 -+ 1.0

14.8 -+ 1.3

Clonidine 15 ug/kg 30 t~g/kg

4 5

> 60 > 60

40.6 -+4.9 a 41.4 ± 3.8b

26.7 -+ 3.5 b 31.4 ± 3.1 a

21.6 -+5.8 26.6 ± 3.1 a

Guanethidine

4

44.6 ± 9.2

25.4 ± 2.1

22.0 -+ 1.5

20.8 + 5.7

Phentolamine 1 mg/kg

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26.1 - 3.7

23.0 ± 3.8

21.1 -+4.7

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28.6 ± 2.1

23.1 ± 3.8

18.9 ± 3.6

a p < 0.05 ; bp < 0.02.

1 V (fig. 2A) and the magnitude of responses at any frequency was not further increased using 6 and 12 V (not shown). 30 min after 30/ag/kg i.v. o f clonidine (solid lines), responses to submaximal stimulation were not importantly affected (fig. 2A), and responses to maximal voltage were slightly reduced (fig. 2B). In contrast, 30 min after guanethidine (0.9 mg/kg i.v.), pronounced inhibition o f responses to 3 V stimulation of the cardiac nerve was observed (fig. 2C). In another group of dogs, the time from the onset of stimulation until the maximum response was reached was measured at various frequencies. The mean values from these experiments plus values obtained in a further group o f dogs, which received phentolamine (1 mg/kg i.v.) prior to clonidine (30 /~g/kg i.v.) are shown in table 1. Compared to pretreatment values, 'clonidine (15 or 30 tag/kg i.v.) markedly extended the duration of stimulation necessary to attain maximum tachycardia at frequencies o f 0.3 and 1.0 Hz. At the lowest frequency, stimulation after clonidine was terminated after 60 sec, at which time there was no increase in heart rate. The effect o f clonidine was less pronounced at the higher frequencies (3 and 10 Hz) of cardiac nerve stimulation. In other dogs, guanethidine~(0.9 mg/kg i.v.) extended the necessary time for maximum tachycardia at 0.3 Hz stimulation but had little or no effect on the time

course at the other frequencies o f nerve stimulation. In a further group of dogs, phentolamine (1 mg/kg i.v.) had no significant effect on any stimulation duration, and a subsequent dose o f clonidine (30 /~g/kg i.v.) failed to extend the duration at frequencies of 0.3 and 1.0 Hz in these dogs.

4. Discussion As shown in table 1, the duration of stimulation necessary to cause maximum tachycardia in response to cardiac nerve stimulation was prolonged by clonidine, particularly at the lower impulse frequencies. Accordingly, the magnitude of response at such frequencies may be markedly reduced by clonidine if the stimulus duration is fixed at some interval below the optimum time. Scriabine and Stavorski (1973) used a fixed 10 sec period o f stimulation which, according to our results, was insufficient to allow maximal tachycardia using frequencies up to 10 Hz in untreated dogs, and was markedly less than the time required at the lower frequencies after 15 and 30 ttg/kg i.v. o f clonidine. Not surprisingly, these workers detected a reduction in the magnitude o f responses at lower frequencies (1.58 and 5.0 Hz), but not at 15.8 Hz, after clonidine. If the stimulus duration was reduced below 10 see, an inhibitory action

R.D. Robson, M.J. Antonaccio, Clonidine and cardiac nerve function

of clonidine was demonstrated at higher frequencies (fig. 1). Both in our earlier (Antonaccio and Robson, 1973) and more recent studies (Antonaccio et al., 1974) in which stimulation was maintained until a maximum response was achieved at each frequency, we did not obtain a significant reduction in magnitude because our experiments did permit an obvious manifestation of the delaying action of clonidine. In the present study, clonidine (30 /ag/kg i.v.) caused negligible depression of the frequency-response curve using optimum stimulus duration and voltage (fig. 2B). Only at 0.3 Hz was there a statistically significant reduction in tachycardia. The importance of stimulus duration is also illustrated in fig. 1 where reductions in responses after clonidine became progressively less as the stimulus duration was increased up to 32 sec at all but the lowest impulse frequency; at 0.3 Hz, stimulation for 32 sec was insufficient to elicit a response (table 1). Guanethidine (0.9 mg/kg i.v.) caused good inhibition of responses at all frequencies of cardiac nerve stimulation (fig. 2C), but caused no significant extension of stimulus duration for maximum response at impulse frequencies of 10, 3 and 1 Hz. Thus, adrenergic neuron blockade after guanethidine was not dependent on stimulus duration at these frequencies. An extension of stimulus duration at 0.3 Hz (table 1) probably was a reflection, to some extent, of the inhibitory action of guanethidine on the maximum response attainable. Nevertheless, if guanethidine should exert a discrete delaying action at this low frequency, it may explain the drug's preferential inhibition of responses to low frequency adrenergic nerve stimulation (Boura and Green, 1962; Green and Robson, 1964). The possibility that a presynaptic a-adrenoceptor mechanism may influence release of adrenergic transmitter was introduced by studies in which a-adrenoceptor agonists and antagonists retard and enhance, respectively, stimulus-evoked release of norepinephrine (Brown and Gillespie, 1957; H~ggendal, 1970; Kirpekar et al., 1972; McCullOch et al., 1972; Starke and Schumann, 1972). Clonidine, which is a powerful a-adrenoceptor stimulant (Hoefke and Kobinger, 1966; Constantine and McShane, 1968), has been shown to inhibit release of norepinephrine from rabbit isolated heart (Starke et al., 1972; Werner et al., 1972), and the effect was prevented by phenoxybenz-

185

amine (Starke and Altmann, 1973). The a-adrenoceptor stimulant action of clonidine also appeared responsible for the extension of stimulus duration necessary to elicit a maximal tachycardia at low impulse frequencies since pretreatment of dogs with phentolamine prevented this action. Possibly, presynaptic a-adrenoceptor stimulation may impose a delay in the release of norepinephrine at low frequencies, but may be unable to retard the more powerful release due to high frequency stimulation.

Acknowledgement The authors would like to thank Mrs. Jeanne Halley and Miss Linda Kerwin for their excellent technical assistance and Boehringer-Ingelheim for the generous supply of clonidine hydrochloride.

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