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Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats
Journal of the Autonomic Nervous System, 20 (1987) 121-127
121
Elsevier
JAN 00743
Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats R.L. Stornetta, P.G. Guyenet and R.C. McCarty Neuroscience Program and Departments of Pharmacology and Psvchology, University of Virginia, Charlottesville, VA 22903 (U.S.A. (Received 3 April 1986) (Revised version received 18 May 1987) (Accepted 19 May 1987)
Key words': Pentobarbital; Urethane; Sympathetic; Parasympathetic; Methyl-atropine; Atenolol Summary Heart rate and blood pressure were recorded in conscious, freely behaving rats through a catheter in the tail artery during administration of nitroprusside or phenylephrine through a catheter in the jugular vein. The sympathetic and parasympathetic components were distinguished by treating the rats with atenolol or methyl-atropine. Reflex bradycardia induced by all doses of phenylephrine was almost totally blocked blocked following methyl-atropine treatment. Reflex tachycardia induced by small to moderate doses of nitroprusside was attenuated to an equal extent following atropine or atenolol treatment. A similar experimental schedule was followed with a separate group of rats to determine the effects of pentobarbital and urethane anesthesia on the baroreceptor reflex. Both pentobarbital and urethane equally attenuated the tachycardia response to a decrease in blood pressure. However, pentobarbital anesthesia resulted in a greater attenuation of the bradycardia response to an increase in blood pressure than did urethane anesthesia. These data support the conclusion that the parasympathetic nervous system is primarily responsible for baroreceptor reflex-induced bradycardia in conscious rats. The sympathetic and parasympathetic systems contribute equally to control baroreceptor reflex-induced tachycardia except in extreme acute hypotension when the tachycardia is predominantly due to the activation of sympathetic nerves. The findings of the second experiment indicate that pentobarbital and urethane affect sympathetic systems differently.
Introduction The relative contributions of sympathetic and parasympathetic input to the baroreceptor reflex and the function of the baroreceptor reflexes in conscious and in anesthetized rats are areas which have not been fully explored. Although these problems have been investigated in other species,
Correspondence: R. Stornetta. Present address: Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22903 U.S.A.
including dogs [2-4,8,16-20,24-26] and rabbits [15,23], the baroreceptor reflex has been studied in rats only under conditions of increased blood pressure [6,7,11]. According to the results of Vatner et al. [26] with dogs, the heart rate changes resulting from changes in blood pressure are the result of different degrees of sympathetic and parasympathetic activity. The present study analyses sympathetic and parasympathetic influences when blood pressure is either increased or decreased to elicit the baroreceptor reflex. Unanesthetized, unrestrained rats and rats with pentobarbital or urethane anesthesia were used.
122
Materials and Methods
carried out one and two days following the catheterization surgery.
General Nine male Sprague-Dawley rats (Hilltop Labs., PA) weighing 300-350 g were anesthetized with sodium pentobarbital (45-55 mg/kg, i.p,) and an indwelling catheter was inserted 2-3 cm up into the ventral tail artery, threaded s.c. and exteriorized at the neck [5]. The jugular vein was cannulated with the tip of the catheter advanced to the atrial sinus and the catheter exteriorized through the same opening in the back of the neck. After recovery from anesthesia, animals were placed individually into clear plastic cages with access to food and water and could move freely about the cage. The catheters were flushed with heparinized saline as necessary to prevent clot formation. Twenty-four hours after the surgery, blood pressure was recorded by attaching the tail artery catheter to a Statham P23D strain gauge transducer connected to a Grass 7-D polygraph. The output from the blood pressure signal was relayed to a tachograph which recorded heart rate from the peak of systole. Mean arterial pressure (MAP) was calculated by hand as: M A P = diastole+ 1/3(systole-diastole).
Baroreceptor reflexes" To elicit baroreceptor reflexes, phenylephrine (L-phenylephrine hydrochloride, Sigma, in 0.9% saline) at doses of (~tg/kg): 0.5, 1, 2, 5 and 10 or sodium nitroprusside (in 0.9% saline) at doses of (~tg/kg): 1, 2, 10, 20 and 50 was administered through the jugular catheter. The doses were given in a 50 #1 bolus followed by a 100 ttl flush of 0.9% saline. The 5 doses of each drug were given in the order: 3rd highest dose, 4th highest dose, lowest dose, 2nd highest dose and highest dose. Heart rate and blood pressure were allowed to return to baseline values before the next dose of a drug was administered. Heart rate and blood pressure were recorded continuously throughout the procedure. To test the reproducibility of cardiovascular responses, a group of 10 rats received injections of nitroprusside (20 t~g/kg) and phenylephrine (5 /tg/kg) while blood pressure and heart rate were monitored as described above. This procedure was
Sympathetic and parasympathetic blockers The sympathetic input to the heart was blocked with the cardio-setective/3 l-antagonist atenolot and the parasympathetic input to the heart was bloCked with atropine methyl-nitrate, a cholinergic antagonist which does not readily cross the blood-brain barrier [12]. After a baseline baroreceptor reflex was established for each animal with the 5 doses of phenylephrine and nitr0prusside, each animal received either atenolol (1 mg/kg, Stuart Pharmaceuticals, Wilmington, DE) or atropine (0.5 mg/kg, atropine methyl nitrate, Sigma) administered through the jugular catheter in a 50 btl bolus followed by a 100 /~1 flush with 0,9% saline. After allowing 5 min for the blocker to take effect, the nitroprusside and phenylephrin e series were repeated. Twenty-four hours later, the procedure was repeated with the other antagonist. The order of administration of atropine and atenolol was randomly determined. All rats included in the analysis received both atropine and atenolot. To test the efficacy of the parasympathetic and sympathetic blockade, a group of 8 rats was tested as described above with injections of nitroprusside (20 /~g/kg) and phenylephrine (5 /~g/kg), first with no drug treatment and then 5 min following simultaneous administration of methyl,atropine (0.5 mg/kg) and atenolol (1 mg/kg).
Anesthetics After a baseline (control) baroreceptor reflex was established with the 5 doses each of phenylephrine and nitroprusside as described above, the animal was anesthetized with sodium pentobarbital (50-55 mg/kg) administered via the jugular catheter. After an adequate level of anesthesia was achieved (assessed by response to hind limb pinch), phenylephrine and nitroprusside were again administered as described above. Twenty-four hours later, the animal was anesthetized with urethane (l.3-1.8 g / k g in 0.9% saline i.p,) until an adequate level of anesthesia was achieved, The administration of nitroprusside a n d phenylephrine was then repeated. (Urethane anesthesia was always given on the second day after surgery because
123 animals did not recover from anesthesia for at least 48 h.)
Data analysis A general linear regression model of analysis which accommodated analysis of variance with repeated measures and covariates was employed to determine the differences between baseline baroreceptor reflex heart rate and blood pressure changes and the responses measured following administration of atropine and atenolol or after pentobarbital and urethane. This method of analysis generates least squares means based on the closest fit regression line in an n-dimensional matrix with appropriate covariates [22]. If baseline heart rate or blood pressure was significantly different between groups, these measures were used as covariates in the analysis of variance for differences between control and drug treatment conditions. If the blood pressure changes induced by phenylephrine or nitroprusside were significantly different between groups, these measures were also used as covariates. The cardiovascular changes induced by nitroprusside and phenylephrine were analyzed separately. All data are presented as mean values _+ S.E.M.
Results
Reproducibility of cardiovascular measures The blood pressure response elicited by nitroprusside (20/.tg/kg) was not significantly different from day ] ( - 4 2 _ + 3 m m H g ) to day 2 ( - 38 + 2 m m Hg). The heart rate response elicited by nitroprusside (20 # g / k g ) was not significantly different from day 1 (106 + 8 b e a t s / m i n ) to day 2 (104 + 9 beats/min). The blood pressure response following phenylephrine (5 ~ g / k g ) was not significantly different from day 1 (51 +_ 3 m m Hg) to day 2 (47_+ 3 m m Hg). Heart rate responses elicited by phenylephrine (5 ~ g / k g ) did not differ from day 1 ( - 1 1 9 _ + 14 beats/rain) to day 2 ( - 107 _+ 7 beats/min). Baseline heart rate and blood pressure for atropine/atenolol group. The resting baseline heart rate of rats prior to drug administration was 348 _+ 9 beats/rain. Resting blood presstire was 110 _
3 m m Hg. The baseline heart rate was decreased by treatment with atenolol to 322 -+ 9 beats/rain ( P = 0.05). The baseline heart rate was increased to 410 -+ 9 b e a t s / m i n ( P < 0.001) after treatment with atropine. The baseline blood pressure was not affected by atenolol (X = 110 + 3 m m Hg) but was significantly increased by atropine (X = 119 _+ 3 mm Hg, P = 0.05). Both baseline blood pressure and heart rate were used as covariates in the analysis of variance to determine differences between control and drug treatment conditions.
Blood pressure and heart rate changes induced by nitroprusside before and after atenolol and atropine administration The efficacy of the sympathetic and parasympathetic blockade for blocking the heart rate response to a dose of 20/.tg/kg of nitroprusside was 86% (15_+4 b e a t s / m i n compared to 108_+9 b e a t s / m i n with no atenolol or atropine treatment, P < 0.01) with no effect on blood pressure change ( - 3 0 _ + 3 m m Hg with combined atropine and atenolol treatment compared with - 3 8 _ + 3 m m Hg for no atenolol or atropine treatment). The heart rate response to 5 ~ g / k g of phenylephrine was virtually eliminated by the combined blockade ( - 1 -+ 2 b e a t s / m i n compared with - 95 -+ 20 b e a t s / m i n for no atenolol or atropine treatment, P < 0.01) with no effect on the blood pressure change (combined atropine/atenolol treatment, 56 _+ 6 m m Hg compared to no treatment, 45 _+ 3 m m Hg). The average decreases in blood pressure induced by 5 doses of nitroprusside before and after administration of either atenolol or atropine are presented in Fig. l. Average blood pressure decreases induced by nitroprusside were slightly but significantly greater after atropine treatment than the decreases seen with either no treatment or atenolol treatment ( F = 59.92, P < 0.0001). Therefore, blood pressure change was used as a covariate in the analysis of variance for heart rate changes after atenolol or atropine treatment. Both atropine and atenolol treatments resulted in a significant reduction of the tachycardia response to nitroprusside injections ( F = 27.19, P < 0.0001) (Fig. 1). Atenolol treatment resulted in a slightly but significantly greater attenuation than
124 150r
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~!~ "10010:1Z01().0;~10~0,00'.I ~01(t,091~.0~.00:1 2~01(~.09fl.050.0 DOSEOF NITROPRUSSIDIE(y.g/kg) Fig. 1. Effects of methyl-atropine or atenolol treatment on baroreceptor reflexes elicited by 5 doses of sodium nitroprusside. GLM means refer to mean values generated by the general linear model analysis of covariance. Changes in heart rate and blood pressure were measured as the difference between peak response following injection of nitroprusside and the resting condition immediately prior to injection. * Differs from corresponding no-pretreatment group, (P < 0.05), • * differs from corresponding methyl atropine pretreatment (P < 0.05) and from no-pretreatment (P < 0.01) groups.
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uHIl 0.5 1.0ZO 5.010.0 0.5 1,02,05.010.0 0.5 1.02.0 5.010.0 DOSEOFPHENYt.,EPHRINE(/~glkgt Fig. 2. Effects of methyl-atropine or atenotol treatment on baroreceptor reflexes elicited by 5 doses of phenyiephrine. GLM means refer to the mean values generated by the general linear model analysis of covariance. Changes in heart rate and blood pressure were measured as the difference between peak response following injection of phenytephrine and the resting condition immediately prior to injection: * Differs from corresponding no-pretreatment and atenotol pretreatment groups ( P < 0.05),
Baseline heart rate and blood pressure Jor the concious / anesthetized group atropine the drop doses of test, P <
of the tachycardia response elicited by in b l o o d pressure at the two highest n i t r o p r u s s i d e ( D u n c a n ' s multiple range 0.05).
Blood pressure and heart rate changes induced 12v phenvlephrine before and after treatment with atenolol or atropine The average b l o o d pressure increases elicited by 5 doses of p h e n y l e p h r i n e b o t h before a n d after t r e a t m e n t with a t r o p i n e or atenolol are depicted in Fig. 2. As was the case with nitroprusside, average b l o o d pressure changes elicited b y p h e n y l e p h r i n e were greater with a t r o p i n e t h a n with atenolol t r e a t m e n t or w i t h o u t either drug p r e t r e a t m e n t ( F = 4.37, P < 0.01). Therefore, b l o o d pressure changes were used as covariates in the analysis of variance of heart rate changes after atenolol or atropine treatment. T h e b r a d y c a r d i a response was severely att e n u a t e d by a t r o p i n e t r e a t m e n t b u t was not c h a n g e d after atenolol t r e a t m e n t ( F = 59.35, P < 0.001) (Fig. 2).
The resting baseline heart rate of conscious rates was 356 + 7 b e a t s / r a i n . Resting blood pressure was 115 ± 4 m m Hg. Baseline heart rate was decreased d u r i n g p e n t o b a r b i t a t anesthesia to 325 7 b e a t s / m i n ( P < 0.001 ~. In contrast, baseline heart rate was increased to 380 + 7 b e a t s / r a i n ( P < 0.05) d u r i n g u r e t h a n e anesthesia. Baseline b l o o d pressure was lowered d u r i n g p e n t o b a r b i t a l anesthesia to 92 z 4 m m H g ~P < 0.05) but was n o t altered by u r e t h a n e 1113 + 4 m m Hg). Both baseline b l o o d pressure a n d heart rate were used as covariates in the analysis of variance to determine differences between conscious and anesthetized conditions.
Blood pressure changes induced by nitroprusside in consctous and pentobarbital- or urethaneanesthetized rats. Average b l o o d pressure decreases i n d u c e d b y nitroprusside were not signific a n t l y different between conscious a n d anesthetized a n i m a l s ( F = 0.26, P = 0.77) (Fig. 3). A d m i n i s t r a t i o n of either p e n t o b a r b i t a l or u r e t h a n e was a t t e n d e d by a stgnificant reduction of the tachycardia response to nitroprusside injec-
125
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Fig. 3. Effects of sodium pentobarbital or urethane anesthesia on baroreceptor reflexes elicited by 5 doses of sodium nitroprusside. * Differs from corresponding no-anesthesia group (P < 0.05).
tions ( F = 173.62, P < 0.0001) as shown in Fig. 3. There was no difference between anesthetics in the degree of attenuation of tachycardia. Blood pressure and heart rate changes induced by phenylephrine in conscious rats and following pentobarbital or urethane anesthesia The blood pressure increases resulting from phenylephrine administration were attenuated during pentobarbital or urethane anesthesia ( F = 13.52, P < 0.0001) (Fig. 4). The blood pressure changes during urethane anesthesia were reduced
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Fig. 4. Effects of sodium pentobarbital or urethane anesthesia on baroreceptor reflexes elicited by 5 doses of phenylephrine. * Differs from corresponding no-anesthesia group ( P < 0.05), * * differs from corresponding no-anesthesia and pentobarbital anesthesia groups (P < 0.05).
to a greater extent than during pentobarbital anesthesia ( P < 0.01). Blood pressure change was used as a covariate in the analysis of variance for differences in heart rate decreases following administration of phenylephrine between conscious and anesthetized states. Fig. 4 shows also that the bradycardia response was severely attenuated during both pentobarbital and urethane anesthesia ( F = 30.05, P < 0.0001). Pentobarbital anesthesia resulted in a greater attenuation of the reflex bradycardia than was evident during urethane anesthesia ( P < 0.0001).
Discussion
One difficulty of the conscious animal preparation is that nerve activity cannot be measured easily; in this study nerve activity is inferred from the responses during pharmacological blockade of autonomic nerves. By administering methyl-atropine and atenolol, the bradycardia response is virtually eliminated. Therefore, the blockers are eliminating the effects of neural activity on baroreceptor-induced bradycardia and no other systems appear to compensate. The combined blockade was effective in eliminating most of the tachycardia response. That some heart rate increase still occurred may indicate either incomplete blockade of neural activity in this instance, or the contribution of a non-fl-adrenergic or noncholinergic system to baroreceptor-induced tachycardia, However, the sympathetic and parasympathetic neural systems are contributing to the great majority of heart rate responses observed. The almost total blockade by atropine of the bradycardic response to increases in blood pressure is in concordance with the results obtained by other workers using anesthetized dogs [13,17,24, 26], rabbits [15], or unanesthetized rats [7]. This indicates a strong parasympathetic component controlling the bradycardia reflex response in conscious rats. The present stu~ly supplies further information on sympathetic versus parasympathetic contributions to baroreceptor-induced tachycardia than previous studies [15,24,26] by utilizing 5 doses of the vasodilator, nitroprusside, to elicit various degrees of tachycardia. The results
126
indicate a balance between sympathetic contributions to the tachycardia response. During smaller heart rate increases, the two systems contribute equally. But during larger heart rate changes, the sympathetic activation predominates. The data from the anesthetized rats support the overall conclusions of other investigators: both pentobarbital [6,8,9,11,14,19,20] and urethane [11,14] severely compromise the baroreceptor reflex. The tachycardia seen after urethane in the present study is consistent with other findings [1,11]. However, there are some discrepancies with other reports on the effects of pentobarbital on baseline heart rate. Other studies have reported an increased heart rate in dogs under pentobarbital anesthesia [14,18]. The present study found a significant bradycardia during pentobarbital anesthesia. This difference is not a species difference, as Fluckinger et al. [11] also reported a tachycardia in rats following pentobarbital. The difference could be in the route of administration as Fluckinger et al. administered the drug i.p. In contrast, the present study used an i.v. injection. The bradycardia we observed might be due to a direct depressant effect of pentobarbital on the myocardium. This effect has been shown in dogs [10,18,21]. The resting heart rate in the study by Fluckinger et al. was also lower than in the current study (295 beats/rain vs 356 beats/min) and this difference might have contributed to the discrepancy in the observations. It is interesting to note that differences in the degree of attenuation of cardiovascular responses did exist between the two anesthetics under conditions of increasing blood pressure (with phenylephrine) and decreasing blood pressure (with nitroprusside). Pentobarbital led to a greater suppression of bradycardia for a given blood pressure increase than did urethane. Given the result that the parasympathetic system contributes more to bradycardia during blood pressure increases, this implies that pentobarbital has a greater depressant effect on parasympathetic tone. This finding is in agreement with the observation by others [25,26] that pentobarbital reduces vagal but not sympathetic tone. Perhaps urethane has the opposite effect of pentobarbital in that it blocks sympathetic tone but leaves parasympathetic tone rel-
atively intact, in agreement with the lesser suppression of bradycardia by urethane than by pentobarbital. Regardless of the specific mechanisms of action of pentobarbital and urethane on sympathetic and parasympathetic contributions to baroreceptor reflex function, it is obvious that these anesthetics disturb normal cardiovascular balance. To obtain representative data on the influence of various experimental manipulations on the sympathetic and the parasympathetic contributions to the baroreceptor reflex, all studies should ideally be performed on unanesthetized animals.
Acknowledgements R.C. McC. was supported by U.S. Public Health Service Grant HL29906 and P.G.G, by U.S. Public Health Service Grant HL28785,
References 1 Adolph. E.F. and Gerbasi. M.J., Blood concentration under influences of Amvtal and urethane. Am J, PhysioL. 106 (19331 35-48, 2 Berkowitz. W.D.. Scherlag, B.J.. Stein. E. and Damato. A.N.. Relative roles of sympathetic and parasympathetic nervous systems in the carotid sinus reflex in dogs, Circ Res',, 24 (19691 447-455. 3 Bouckaert. J.J. and Heymans, (The influence o) barbiturates on the proprioceptive mechanisms of vaso-mofor tone regulation. J. Physiol. (London), 90 (1937) 59P-60P. 4 Brown, R.V. and Hilton. J.G., The effectiveness of the baroreceptor reflexes under different anesthetics. J. Pharmacol. Exp, Ther,. 118 (1956) 198-203. 5 Chiueh, C.C. and Kopin, I.J., Hyperresponsivity of spontaneously hypertensive rats to indirect measurement of blood pressure. Am. J. Physiol., 234 (1978) H690-695. 6 Coleman. T.G., Bengls, R.G. and Samar. R.E,. Baroreflexes and anesthesia in the rat. PhysiolOgist, 20 (1977) 18. 7 Coleman. T.G.. Arterial baroreflexcontrol of heart rate in the conscious rat, Am. J. Physiol., 238 (t980] H515-520. 8 Cox. R.H. and Bagshaw, R.J.. Influence of anesthesia on the response to carotid hypotension in dogs, Am. J. Physiol., 237 (1979) H424-432. 9 Cox. R.H. and Bagshaw. J., Effects of anesthesia on carotid sinus reflex control of arterial hemodynarmcs in the dog, Am, .L Physiol., 239 ( 19801 H681-69t. 10 Daniel, E.E.. Fulton, J.B., Hiddleston. M,. Martin, W. and Foulks. J.G., An analysis of the mechanism of barbiturate induced cardiovascular depression and its antagonism by
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12
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14
15
16
17
sympathomimetic amines, Arch. Int. Pharmacody., 108 (1956) 457 472. Fluckinger, J-P., Sonnay, M. Boillat, N. and Atkinson, J., Attenuation of the baroreceptor reflex by general anesthetic agents in the normotensive rat, Eur. J. Pharmacol., 109 (1985) 105-109. Gilman, A.G,, Goodman, L.S. and Gilman, A., The Pharmacological Basis of Therapeutics, McMillan, New York, 1980. Glick, G. and Braunwald, E., Relative roles of the sympathetic and parasympathetic nervous systems in the reflex control of heart rate, Circ. Res., 16 (1965) 363-375. Greisheimer, E.M., The circulatory effects of anesthetics. In: Handbook of Physiology, Circulation, Section 2, Vol. Ill, American Physiological Society, Washington, D.C., 1965, pp. 2477 2510. Guo, G.B. Thames, M.D. and Abboud, F.M., Differential baroreflex control of heart rate and vascular resistance in rabbits, Ctrc. Res., 50 (1982) 554-565. lriuchijima, J., Soulsby, M.E. and Wilson, M.F., Participation of cardiac sympathetics in carotid occlusion pressor reflex, Am. J. Physiol., 215 (1968) 1111-1114. Kollai, M. and Koizumi, K., Reciprocal and non-reciprocal action of the vagal and sympathetic nerves innervating the heart, ,1. Auton. Nero. Syst., 1 (1979) 33 52.
18 Nash, C.B., Davis, F.and Woodbury, R.A., Cardiovascular effects of anesthetic doses of pentobarbital sodium, Am. J. Pt~vsiol., 185 (1956) 107-112. 19 Peiss, C.N. and Manning, J.W., Effects of sodium pentobarbital on electrical and reflex activation of the cardiovascular system, Circ. Res., 14 (1964) 228 235. 20 Price, H.L., General anesthesia and circulatory homeostatis, Physiol. Reu., 40 (1960) 187-218. 21 Roth, G.B., Barbiturates on the mammalian heart, Arch. Int. Pharrnacodyn., 51 (1935) 179-184. 22 Searle, S.R., Linear Models, John Wiley, New York, 1971. 23 Stinnett, H.O., Sepe, F.J. and Mangusson, M.R., Rabbit carotid baroreflexes after carotid sympathectomy, vagotomy and B blockade, Am. J. Physiol., 231 (198l) H600-605. 24 Thames, M.D. and Kontos, H.A., Mechanisms of baroreceptor-induced changes in heart rate, Am. J. Pl~vsiol., 218 (1970) 251-256. 25 Vatner, S.F., Franklin, D. and Braunwald, E., Effects of anesthesia and sleep on circulatory response to carotid sinus nerve stimulation, Am. J. Physiol., 220 (1971) 1249-1255. 26 Vamer, S.F., Higgins, C.B. and Braunwald, E., Sympathetic and parasympathetic components of reflex tachycardia induced by hypotension in conscious dogs with and without heart failure, Cardiovasc. Res., 8 (1974) 153-161.
121
Elsevier
JAN 00743
Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats R.L. Stornetta, P.G. Guyenet and R.C. McCarty Neuroscience Program and Departments of Pharmacology and Psvchology, University of Virginia, Charlottesville, VA 22903 (U.S.A. (Received 3 April 1986) (Revised version received 18 May 1987) (Accepted 19 May 1987)
Key words': Pentobarbital; Urethane; Sympathetic; Parasympathetic; Methyl-atropine; Atenolol Summary Heart rate and blood pressure were recorded in conscious, freely behaving rats through a catheter in the tail artery during administration of nitroprusside or phenylephrine through a catheter in the jugular vein. The sympathetic and parasympathetic components were distinguished by treating the rats with atenolol or methyl-atropine. Reflex bradycardia induced by all doses of phenylephrine was almost totally blocked blocked following methyl-atropine treatment. Reflex tachycardia induced by small to moderate doses of nitroprusside was attenuated to an equal extent following atropine or atenolol treatment. A similar experimental schedule was followed with a separate group of rats to determine the effects of pentobarbital and urethane anesthesia on the baroreceptor reflex. Both pentobarbital and urethane equally attenuated the tachycardia response to a decrease in blood pressure. However, pentobarbital anesthesia resulted in a greater attenuation of the bradycardia response to an increase in blood pressure than did urethane anesthesia. These data support the conclusion that the parasympathetic nervous system is primarily responsible for baroreceptor reflex-induced bradycardia in conscious rats. The sympathetic and parasympathetic systems contribute equally to control baroreceptor reflex-induced tachycardia except in extreme acute hypotension when the tachycardia is predominantly due to the activation of sympathetic nerves. The findings of the second experiment indicate that pentobarbital and urethane affect sympathetic systems differently.
Introduction The relative contributions of sympathetic and parasympathetic input to the baroreceptor reflex and the function of the baroreceptor reflexes in conscious and in anesthetized rats are areas which have not been fully explored. Although these problems have been investigated in other species,
Correspondence: R. Stornetta. Present address: Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22903 U.S.A.
including dogs [2-4,8,16-20,24-26] and rabbits [15,23], the baroreceptor reflex has been studied in rats only under conditions of increased blood pressure [6,7,11]. According to the results of Vatner et al. [26] with dogs, the heart rate changes resulting from changes in blood pressure are the result of different degrees of sympathetic and parasympathetic activity. The present study analyses sympathetic and parasympathetic influences when blood pressure is either increased or decreased to elicit the baroreceptor reflex. Unanesthetized, unrestrained rats and rats with pentobarbital or urethane anesthesia were used.
122
Materials and Methods
carried out one and two days following the catheterization surgery.
General Nine male Sprague-Dawley rats (Hilltop Labs., PA) weighing 300-350 g were anesthetized with sodium pentobarbital (45-55 mg/kg, i.p,) and an indwelling catheter was inserted 2-3 cm up into the ventral tail artery, threaded s.c. and exteriorized at the neck [5]. The jugular vein was cannulated with the tip of the catheter advanced to the atrial sinus and the catheter exteriorized through the same opening in the back of the neck. After recovery from anesthesia, animals were placed individually into clear plastic cages with access to food and water and could move freely about the cage. The catheters were flushed with heparinized saline as necessary to prevent clot formation. Twenty-four hours after the surgery, blood pressure was recorded by attaching the tail artery catheter to a Statham P23D strain gauge transducer connected to a Grass 7-D polygraph. The output from the blood pressure signal was relayed to a tachograph which recorded heart rate from the peak of systole. Mean arterial pressure (MAP) was calculated by hand as: M A P = diastole+ 1/3(systole-diastole).
Baroreceptor reflexes" To elicit baroreceptor reflexes, phenylephrine (L-phenylephrine hydrochloride, Sigma, in 0.9% saline) at doses of (~tg/kg): 0.5, 1, 2, 5 and 10 or sodium nitroprusside (in 0.9% saline) at doses of (~tg/kg): 1, 2, 10, 20 and 50 was administered through the jugular catheter. The doses were given in a 50 #1 bolus followed by a 100 ttl flush of 0.9% saline. The 5 doses of each drug were given in the order: 3rd highest dose, 4th highest dose, lowest dose, 2nd highest dose and highest dose. Heart rate and blood pressure were allowed to return to baseline values before the next dose of a drug was administered. Heart rate and blood pressure were recorded continuously throughout the procedure. To test the reproducibility of cardiovascular responses, a group of 10 rats received injections of nitroprusside (20 t~g/kg) and phenylephrine (5 /tg/kg) while blood pressure and heart rate were monitored as described above. This procedure was
Sympathetic and parasympathetic blockers The sympathetic input to the heart was blocked with the cardio-setective/3 l-antagonist atenolot and the parasympathetic input to the heart was bloCked with atropine methyl-nitrate, a cholinergic antagonist which does not readily cross the blood-brain barrier [12]. After a baseline baroreceptor reflex was established for each animal with the 5 doses of phenylephrine and nitr0prusside, each animal received either atenolol (1 mg/kg, Stuart Pharmaceuticals, Wilmington, DE) or atropine (0.5 mg/kg, atropine methyl nitrate, Sigma) administered through the jugular catheter in a 50 btl bolus followed by a 100 /~1 flush with 0,9% saline. After allowing 5 min for the blocker to take effect, the nitroprusside and phenylephrin e series were repeated. Twenty-four hours later, the procedure was repeated with the other antagonist. The order of administration of atropine and atenolol was randomly determined. All rats included in the analysis received both atropine and atenolot. To test the efficacy of the parasympathetic and sympathetic blockade, a group of 8 rats was tested as described above with injections of nitroprusside (20 /~g/kg) and phenylephrine (5 /~g/kg), first with no drug treatment and then 5 min following simultaneous administration of methyl,atropine (0.5 mg/kg) and atenolol (1 mg/kg).
Anesthetics After a baseline (control) baroreceptor reflex was established with the 5 doses each of phenylephrine and nitroprusside as described above, the animal was anesthetized with sodium pentobarbital (50-55 mg/kg) administered via the jugular catheter. After an adequate level of anesthesia was achieved (assessed by response to hind limb pinch), phenylephrine and nitroprusside were again administered as described above. Twenty-four hours later, the animal was anesthetized with urethane (l.3-1.8 g / k g in 0.9% saline i.p,) until an adequate level of anesthesia was achieved, The administration of nitroprusside a n d phenylephrine was then repeated. (Urethane anesthesia was always given on the second day after surgery because
123 animals did not recover from anesthesia for at least 48 h.)
Data analysis A general linear regression model of analysis which accommodated analysis of variance with repeated measures and covariates was employed to determine the differences between baseline baroreceptor reflex heart rate and blood pressure changes and the responses measured following administration of atropine and atenolol or after pentobarbital and urethane. This method of analysis generates least squares means based on the closest fit regression line in an n-dimensional matrix with appropriate covariates [22]. If baseline heart rate or blood pressure was significantly different between groups, these measures were used as covariates in the analysis of variance for differences between control and drug treatment conditions. If the blood pressure changes induced by phenylephrine or nitroprusside were significantly different between groups, these measures were also used as covariates. The cardiovascular changes induced by nitroprusside and phenylephrine were analyzed separately. All data are presented as mean values _+ S.E.M.
Results
Reproducibility of cardiovascular measures The blood pressure response elicited by nitroprusside (20/.tg/kg) was not significantly different from day ] ( - 4 2 _ + 3 m m H g ) to day 2 ( - 38 + 2 m m Hg). The heart rate response elicited by nitroprusside (20 # g / k g ) was not significantly different from day 1 (106 + 8 b e a t s / m i n ) to day 2 (104 + 9 beats/min). The blood pressure response following phenylephrine (5 ~ g / k g ) was not significantly different from day 1 (51 +_ 3 m m Hg) to day 2 (47_+ 3 m m Hg). Heart rate responses elicited by phenylephrine (5 ~ g / k g ) did not differ from day 1 ( - 1 1 9 _ + 14 beats/rain) to day 2 ( - 107 _+ 7 beats/min). Baseline heart rate and blood pressure for atropine/atenolol group. The resting baseline heart rate of rats prior to drug administration was 348 _+ 9 beats/rain. Resting blood presstire was 110 _
3 m m Hg. The baseline heart rate was decreased by treatment with atenolol to 322 -+ 9 beats/rain ( P = 0.05). The baseline heart rate was increased to 410 -+ 9 b e a t s / m i n ( P < 0.001) after treatment with atropine. The baseline blood pressure was not affected by atenolol (X = 110 + 3 m m Hg) but was significantly increased by atropine (X = 119 _+ 3 mm Hg, P = 0.05). Both baseline blood pressure and heart rate were used as covariates in the analysis of variance to determine differences between control and drug treatment conditions.
Blood pressure and heart rate changes induced by nitroprusside before and after atenolol and atropine administration The efficacy of the sympathetic and parasympathetic blockade for blocking the heart rate response to a dose of 20/.tg/kg of nitroprusside was 86% (15_+4 b e a t s / m i n compared to 108_+9 b e a t s / m i n with no atenolol or atropine treatment, P < 0.01) with no effect on blood pressure change ( - 3 0 _ + 3 m m Hg with combined atropine and atenolol treatment compared with - 3 8 _ + 3 m m Hg for no atenolol or atropine treatment). The heart rate response to 5 ~ g / k g of phenylephrine was virtually eliminated by the combined blockade ( - 1 -+ 2 b e a t s / m i n compared with - 95 -+ 20 b e a t s / m i n for no atenolol or atropine treatment, P < 0.01) with no effect on the blood pressure change (combined atropine/atenolol treatment, 56 _+ 6 m m Hg compared to no treatment, 45 _+ 3 m m Hg). The average decreases in blood pressure induced by 5 doses of nitroprusside before and after administration of either atenolol or atropine are presented in Fig. l. Average blood pressure decreases induced by nitroprusside were slightly but significantly greater after atropine treatment than the decreases seen with either no treatment or atenolol treatment ( F = 59.92, P < 0.0001). Therefore, blood pressure change was used as a covariate in the analysis of variance for heart rate changes after atenolol or atropine treatment. Both atropine and atenolol treatments resulted in a significant reduction of the tachycardia response to nitroprusside injections ( F = 27.19, P < 0.0001) (Fig. 1). Atenolol treatment resulted in a slightly but significantly greater attenuation than
124 150r
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of the tachycardia response elicited by in b l o o d pressure at the two highest n i t r o p r u s s i d e ( D u n c a n ' s multiple range 0.05).
Blood pressure and heart rate changes induced 12v phenvlephrine before and after treatment with atenolol or atropine The average b l o o d pressure increases elicited by 5 doses of p h e n y l e p h r i n e b o t h before a n d after t r e a t m e n t with a t r o p i n e or atenolol are depicted in Fig. 2. As was the case with nitroprusside, average b l o o d pressure changes elicited b y p h e n y l e p h r i n e were greater with a t r o p i n e t h a n with atenolol t r e a t m e n t or w i t h o u t either drug p r e t r e a t m e n t ( F = 4.37, P < 0.01). Therefore, b l o o d pressure changes were used as covariates in the analysis of variance of heart rate changes after atenolol or atropine treatment. T h e b r a d y c a r d i a response was severely att e n u a t e d by a t r o p i n e t r e a t m e n t b u t was not c h a n g e d after atenolol t r e a t m e n t ( F = 59.35, P < 0.001) (Fig. 2).
The resting baseline heart rate of conscious rates was 356 + 7 b e a t s / r a i n . Resting blood pressure was 115 ± 4 m m Hg. Baseline heart rate was decreased d u r i n g p e n t o b a r b i t a t anesthesia to 325 7 b e a t s / m i n ( P < 0.001 ~. In contrast, baseline heart rate was increased to 380 + 7 b e a t s / r a i n ( P < 0.05) d u r i n g u r e t h a n e anesthesia. Baseline b l o o d pressure was lowered d u r i n g p e n t o b a r b i t a l anesthesia to 92 z 4 m m H g ~P < 0.05) but was n o t altered by u r e t h a n e 1113 + 4 m m Hg). Both baseline b l o o d pressure a n d heart rate were used as covariates in the analysis of variance to determine differences between conscious and anesthetized conditions.
Blood pressure changes induced by nitroprusside in consctous and pentobarbital- or urethaneanesthetized rats. Average b l o o d pressure decreases i n d u c e d b y nitroprusside were not signific a n t l y different between conscious a n d anesthetized a n i m a l s ( F = 0.26, P = 0.77) (Fig. 3). A d m i n i s t r a t i o n of either p e n t o b a r b i t a l or u r e t h a n e was a t t e n d e d by a stgnificant reduction of the tachycardia response to nitroprusside injec-
125
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Fig. 3. Effects of sodium pentobarbital or urethane anesthesia on baroreceptor reflexes elicited by 5 doses of sodium nitroprusside. * Differs from corresponding no-anesthesia group (P < 0.05).
tions ( F = 173.62, P < 0.0001) as shown in Fig. 3. There was no difference between anesthetics in the degree of attenuation of tachycardia. Blood pressure and heart rate changes induced by phenylephrine in conscious rats and following pentobarbital or urethane anesthesia The blood pressure increases resulting from phenylephrine administration were attenuated during pentobarbital or urethane anesthesia ( F = 13.52, P < 0.0001) (Fig. 4). The blood pressure changes during urethane anesthesia were reduced
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to a greater extent than during pentobarbital anesthesia ( P < 0.01). Blood pressure change was used as a covariate in the analysis of variance for differences in heart rate decreases following administration of phenylephrine between conscious and anesthetized states. Fig. 4 shows also that the bradycardia response was severely attenuated during both pentobarbital and urethane anesthesia ( F = 30.05, P < 0.0001). Pentobarbital anesthesia resulted in a greater attenuation of the reflex bradycardia than was evident during urethane anesthesia ( P < 0.0001).
Discussion
One difficulty of the conscious animal preparation is that nerve activity cannot be measured easily; in this study nerve activity is inferred from the responses during pharmacological blockade of autonomic nerves. By administering methyl-atropine and atenolol, the bradycardia response is virtually eliminated. Therefore, the blockers are eliminating the effects of neural activity on baroreceptor-induced bradycardia and no other systems appear to compensate. The combined blockade was effective in eliminating most of the tachycardia response. That some heart rate increase still occurred may indicate either incomplete blockade of neural activity in this instance, or the contribution of a non-fl-adrenergic or noncholinergic system to baroreceptor-induced tachycardia, However, the sympathetic and parasympathetic neural systems are contributing to the great majority of heart rate responses observed. The almost total blockade by atropine of the bradycardic response to increases in blood pressure is in concordance with the results obtained by other workers using anesthetized dogs [13,17,24, 26], rabbits [15], or unanesthetized rats [7]. This indicates a strong parasympathetic component controlling the bradycardia reflex response in conscious rats. The present stu~ly supplies further information on sympathetic versus parasympathetic contributions to baroreceptor-induced tachycardia than previous studies [15,24,26] by utilizing 5 doses of the vasodilator, nitroprusside, to elicit various degrees of tachycardia. The results
126
indicate a balance between sympathetic contributions to the tachycardia response. During smaller heart rate increases, the two systems contribute equally. But during larger heart rate changes, the sympathetic activation predominates. The data from the anesthetized rats support the overall conclusions of other investigators: both pentobarbital [6,8,9,11,14,19,20] and urethane [11,14] severely compromise the baroreceptor reflex. The tachycardia seen after urethane in the present study is consistent with other findings [1,11]. However, there are some discrepancies with other reports on the effects of pentobarbital on baseline heart rate. Other studies have reported an increased heart rate in dogs under pentobarbital anesthesia [14,18]. The present study found a significant bradycardia during pentobarbital anesthesia. This difference is not a species difference, as Fluckinger et al. [11] also reported a tachycardia in rats following pentobarbital. The difference could be in the route of administration as Fluckinger et al. administered the drug i.p. In contrast, the present study used an i.v. injection. The bradycardia we observed might be due to a direct depressant effect of pentobarbital on the myocardium. This effect has been shown in dogs [10,18,21]. The resting heart rate in the study by Fluckinger et al. was also lower than in the current study (295 beats/rain vs 356 beats/min) and this difference might have contributed to the discrepancy in the observations. It is interesting to note that differences in the degree of attenuation of cardiovascular responses did exist between the two anesthetics under conditions of increasing blood pressure (with phenylephrine) and decreasing blood pressure (with nitroprusside). Pentobarbital led to a greater suppression of bradycardia for a given blood pressure increase than did urethane. Given the result that the parasympathetic system contributes more to bradycardia during blood pressure increases, this implies that pentobarbital has a greater depressant effect on parasympathetic tone. This finding is in agreement with the observation by others [25,26] that pentobarbital reduces vagal but not sympathetic tone. Perhaps urethane has the opposite effect of pentobarbital in that it blocks sympathetic tone but leaves parasympathetic tone rel-
atively intact, in agreement with the lesser suppression of bradycardia by urethane than by pentobarbital. Regardless of the specific mechanisms of action of pentobarbital and urethane on sympathetic and parasympathetic contributions to baroreceptor reflex function, it is obvious that these anesthetics disturb normal cardiovascular balance. To obtain representative data on the influence of various experimental manipulations on the sympathetic and the parasympathetic contributions to the baroreceptor reflex, all studies should ideally be performed on unanesthetized animals.
Acknowledgements R.C. McC. was supported by U.S. Public Health Service Grant HL29906 and P.G.G, by U.S. Public Health Service Grant HL28785,
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