Experimental Gerontology, Vol. 26, pp. 65-75, 1991 Printedin the USA. All rightsreserved.
0531-5565/91 $3.00 + .00 Copyright©1991 PergamonPress plc
AUTONOMIC REGULATION OF REFLEX BRADYCARDIA IN RATS DECLINES WITH AGE
DONNA L. BARRINGERand RUBEN D. BU/qAG Department of Pharmacology, College of Health Sciences and Hospital, Universityof Kansas Medical Center, Kansas City, KS 66103
A b s t r a c t - Heart rate responses, elicited reflexly by elevating blood pressure with phenylephrine or lowering it with sodium nitroprusside, were compared in groups of rats aged 4 and 14 months. All tests were done while the rats were awake to avoid artefacts due to anesthesia. Parasympathetic and sympathetic contributions were assessed by repeating baroreflex tests after cholinergic blockade with atropine or 13-adrenergic blockade with propranolol. Magnitude of reflex bradycardia and tachycardia was initially smaller in the 14-month-old rats than in the younger rats thereby indicating that baroreflex sensitivity had diminished with age. After 13-adrenergic or cholinergic blockade, adjusted means (obtained by covariance analysis) for reflex tachycardia did not differ significantly between rat groups, but those for reflex bradycardia were significantly smaller in the 14-month-old rats than in the younger rats. Selective attenuation of reflex bradycardia after either cholinergic or 13adrenergic blockade indicates that concurrent parasympathetic activation and sympathetic withdrawal during reflex bradycardia were also reduced in 14-month-old rats. These results suggest that as autonomic mediation of reflex bradycardia diminishes with age, old rats may no longer be able to slow the heart as easily whenever blood pressure rises, but they can still accelerate it whenever blood pressure falls. Key Words: aging, baroreflexes, ~-adrenergic blockade, blood pressure, conscious rats, cholinergic blockade, heart rate, parasympathetic, sympathetic
INTRODUCTION BAROREFLEX ATI'ENUATIONin old rats has been described previously (Docherty et al., 1986; Rothbaum et al., 1974; Tanabe and Bufiag, 1989), but underlying autonomic mechanisms remain uncertain. Of the studies that have used selective pharmacological blockade to identify sympathetic and parasympathetic contributions to heart rate reflexes in dogs (Berkowitz et al., 1969; Glick and Braunwald, 1965; Scher and Young, 1970; Vatner et al., 1974) and rats (Ferrari et al. 1987; Rothbaum et al., 1974; Stornetta et al., 1987), only one has described an
Correspondence to: Ruben D. Bufiag, Departmentof Pharmacology,Universityof Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS 66103. (Received 22 March 1990; Accepted 1 October 1990) 65
66
D. L. BARRINGER and R. D. BU/qAG
age-related attenuation of vagal tone during reflex bradycardia (Rothbaum et al., 1974) and possible changes in attendant sympathetic mediation were not explored. In anesthetized 9-month rats, Tanabe and Bufiag (1989) recently showed that while baroreflex sensitivity was still adequate at normotensive pressures, it was already impaired at pressures above 130 mm Hg. Whether a similar age-related baroreflex impairment would be demonstrable in conscious rats is unknown. Accordingly, in the present studies we compared reflex heart rate responses in conscious 4-month and 14-month-old rats to determine whether sympathetic and parasympathetic contributions to heart rate control change with age. Reflex heart rate responses were elicited by elevating blood pressure with phenylephrine or lowering it with sodium nitroprusside. Cholinergic and [3-adrenergic contributions were later quantified by using atropine and propranolol to block efferent parasympathetic and sympathetic pathways, respectively. MATERIALS AND METHODS Ten 4-month-old and thirteen 14-month-old female Sprague-Dawley rats were purchased from SASCO Inc (Omaha, NE). All rats were transiently anesthetized for surgical implantation of indwelling vascular cannulas. One week later, heart rates were recorded as blood pressure was altered by infusing phenylephrine or sodium nitroprusside intravenously while the rats were awake. For baroreflex testing, drugs were infused five times on postoperative days 7 and 8. On both days, baroreflex tests were first done prior to antagonist pretreatment and then repeated following cholinergic or 13-adrenergic blockade. Additionally, on day 8, a third test was done following combined cholinergic and 13-adrenergic blockade.
Surgical preparation for baroreflex testing After anesthetizing each rat by intraperitoneal injection of a mixture of ketamine (10 mg/ 100 g as a dissociative anesthetic) and acepromazine (0.1 mg/100 g as a supplementary tranquilizer), indwelling cannulas were inserted separately into the right femoral artery for recording blood pressure and the ipsilateral vein for infusing drugs (Barringer and Bufiag, 1989; Bufiag and Miyajima, 1984). Cannulas consisted of two pieces of polyethylene tubing (a 2-4 cm length of PE-10 threaded and heat-fused into a 13-18 cm length of PE-50 tubing) with a total dead-space volume of 0.05-0.1 ml. The end with PE-10 tubing was inserted directly into the blood vessel while the larger bore PE-50 tubing was passed subcutaneously to emerge at the nape. To keep cannulas patent, immediately after surgical implantation and on the third and fifth postoperative days, they were cleared by rapidly injecting 0.5 ml saline, then filled with heparinized (30 U/ml) saline. Following surgery and between recording sessions, rats were kept in individual cages in an air-conditioned room with unrestricted access to food (Rodent Laboratory Chow, Ralston Purina Company, St. Louis, MO) and water.
Recording reflex chronotropic responses to intravenously-infused drugs One week after surgical cannulation, with each awake rat kept in its own open-topped plastic cage, pulsatile pressure was recorded on a Gould polygraph by connecting the arterial cannula through PE-50 tubing to a small-volume-displacement pressure transducer (PI0EZ from Spectramed Inc, Critical Care Division, Oxnard, CA) placed at the same level as the rat. Heart
AGE-RELATED BAROREFLEX A'I'rENUATION
67
rate was monitored simultaneously by triggering a biotachometer with the phasic pressure signal from the transducer. The venous cannula was connected to a syringe pump (Harvard Apparatus, South Natick, MA) for drug infusions. After connecting both cannulas, 45--60 min was allowed to lapse without further manipulation so that the rat could become accustomed to the recording environment. Arterial pressures and heart rates were then recorded continuously during slow intravenous infusions, first of phenylephrine and then of sodium nitroprusside. Doses (nanograms salt/100 grams body weight/min) ranging from 750 to 1500 for L-phenylephrine hydrochloride and 550 to 1100 for sodium nitroprusside were infused at a rate of 20 Ixl/min for 30 seconds using a syringe pump. All infusions were regulated to alter arterial pressure progressively by 30-40 mm Hg within 30 seconds. Phenylephrine was diluted with isotonic saline while sodium nitroprusside was prepared in 5% dextrose and protected from light to retard spontaneous decay. Reflex heart rate responses to drug-induced changes in arterial pressure were recorded five times on two successive days. On the seventh day after cannulation, a control recording was taken first, and then followed by a second recording 15 min after either cholinergic blockade with atropine sulfate, or B-blockade with propranolol hydrochloride; doses of 0.1 mg/100 g IV used for both drugs have previously been found effective in abolishing all heart rate responses elicited reflexly by phenylephrine or sodium nitroprusside (Barringer and Bufiag, 1989; Coleman, 1980). On the eighth day, baroreflex responses were recorded three more times: (1) an initial test before antagonist pretreatment, (2) a second test 15 min after blockade with either atropine or propranolol (using the antagonist that had not been used the previous day), and (3) a third test 15 min after combined blockade induced by added injection of the second antagonist.
Data analysis and statistics All data (except adjusted means) were expressed as averages + SEM. Mean arterial pressure was calculated as diastolic pressure plus one-third of the pulse pressure. Baroreflex responses during infusions of phenylephrine or sodium nitroprusside were analyzed by reading heart rate values at each 5 mm Hg step-wise change in mean arterial pressure (up to 30 mm Hg). Differences in reflex heart rate changes between young and old groups were assessed using a repeated measures analysis of variance. Mainly because initial reflex respoffses differed between age groups, and also because baseline heart rates were altered significantly following treatment with atropine or propranolol, reflex responsiveness following antagonist treatment was assessed by covariance analysis using data obtained after blockade as the dependent variable and data recorded from the same rats prior to blockade as the covariate. Whenever F-Ratios were significant, Newman-Keuls' multiple range test (Bruning and Kintz, 1987) was used to determine significance between pairs of means. P-values of 0.05 or less were considered statistically significant. Repeated measures analysis of covariance was done on an IBM Model 4381 using BMDP Statistical Software (BMDP, 1985), while all other statistical tests were done on an IBM XT computer with the Number Cruncher Statistical System (Hintze, 1986). RESULTS
Stability of reflex responsiveness in conscious rats In all rats, intravenous infusions of phenylephrine gradually elevated mean arterial pressure and reflexly slowed heart rate (Figure 1) while similar infusions of sodium nitroprusside had
68
D. L. BARRINGER and R D. BUIqAG
A
B
200-~
Fig. I. Changes in pulsatile pressure (mm Hg, lower panel) and heart rate (bpm, upper panel) during continuous phenylephrine infusion in a 4-month-old (A) and 14-month-old(B) rat. Chart speed 125 mm/min. Arrow indicates beginning of infusion. opposite effects of gradually lowering mean arterial pressure and reflexly increasing heart rate (Figure 2). Blood pressure rose or fell by 5 - 8 mm Hg per 5 seconds and with either drug, magnitude of the reflex heart rate response increased as the corresponding change in mean pressure became greater. Drug doses required to lower or elevate blood pressure by 30 to 40 mm Hg were similar regardless of age. Within each rat group, initial chronotropic responses to increases or decreases in blood pressure were qualitatively and quantitatively the same on days 7 and 8. The absence of significant differences between days indicated that antagonists administered on the first day were no longer present on the second day so that effects of another antagonist could be tested. Since it also shows that reflex responsiveness was reproducible from day to day, results from both days were pooled (Table l) to obtain single values representing average reflex responses prior to antagonist treatment.
A
B
Fig. 2. Changes in pulsatile pressure (mm Hg, lower panel) and heart rate (bpm, upper panel) during continuous sodium nitroprusside infusion in a 4-month-old (A) and 14-month-old(B) rat. Chart speed 125 mm/min. Arrow indicates beginning of infusion.
69
AGE-RELATED BAROREFLEX A'ITENUAT1ON TABLE 1. AVERAGE REFLEX HEART RATE RESPONSES ( b p m ) ELICITED BY INTRAVENOUSLYINFUSEDDRUGS IN CONSCIOUS 4-MONTH AND 14-MONTH OLD RATS*
Age Group
5
10
Change in Mean Pressure (Am Hg) 15 20
25
30
-51 -+ 4 -31 + 5a
- 5 9 -4- 5 - 3 5 --- 5a
66 --- 9 40 + 5a
79 --- 10 45 -+ 7a
(A) Reflex Bradycardia to Phenylephrine 4-month 14-month
- 1 0 -- 2 - 6 --- 2
- 1 9 --- 2 - 1 3 -4- 2
- 3 2 --- 5 -21 +- 4a
- 4 2 --- 5 - 2 7 + 4a
F-ratio = 10.75, p < 0.004
(B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
9 +-- 3 3 --- 1
21 --- 4 12 +-- 2
35 +-- 5 23 --- 3
48 --- 6 32 -+- 4 a
F-ratio = 10.45, p < 0.004 *Expressed as averages ___ SEM from 10 rats in each group by combining data on days 7 and 8. ap < 0.05 as compared with corresponding average for 4-month-old rats using Newman-Keuls' multiple range test.
Baroreflex attenuation in 14-month old rats M a g n i t u d e o f either reflex bradycardia or tachycardia was generally smaller in 14-month than in 4 - m o n t h - o l d rats. F o r reflex bradycardia p r o d u c e d during p h e n y l e p h r i n e - i n d u c e d pressor responses o f 15 m m Hg or m o r e , or for reflex tachycardia p r o d u c e d during s o d i u m nitroprusside induced depressor responses o f - 2 0 m m H g or m o r e (Table 1) the differences b e t w e e n rat groups w e r e significant. By indicating that baroreflex sensitivity, whether manifested as bradycardia or tachycardia, was attenuated in the older rats this finding confirms the age-related baroreflex i m p a i r m e n t reported p r e v i o u s l y by T a n a b e and Bufiag (1989). B e c a u s e heart rate reflexes n o r m a l l y d e p e n d on efferent parasympathetic and sympathetic pathways, it was considered possible that reduced baroreflex sensitivity in old rats m a y reflect an a u t o n o m i c imbalance. This possibility was tested by recording reflex responses in the same rats after appropriate p h a r m a c o l o g i c a l blockade: [3-adrenergic blockade with propranolol to reveal parasympathetic m e c h a n i s m s , or cholinergic blockade with atropine to reveal sympathetic m e c h a n i s m s .
Selective autonomic blockade altered basal heart rate but not blood pressure Initial baselines for either m e a n pressure or heart rate did not differ significantly b e t w e e n age groups (Table 2). Basal heart rates w e r e elevated f o l l o w i n g cholinergic b l o c k a d e with atropine and l o w e r e d f o l l o w i n g [3-adrenergic b l o c k a d e with propranolol, while those for m e a n pressure w e r e unaffected (Table 2). D i f f e r e n c e s b e t w e e n age groups in ensuing heart rate changes w e r e not significant; cholinergic b l o c k a d e increased baseline heart rates (bpm) by 55 +_ 11 in 4 - m o n t h - o l d rats and 36 ___ 8 in 14-month-old rats, while [3-adrenergic blockade decreased baseline heart rates by 51 --- 6 and 56 --- 8, respectively. Regardless o f age, heart rates f o l l o w i n g c o m b i n e d b l o c k a d e with both atropine and propranolol w e r e significantly l o w e r than the original basal levels (Table 2).
70
D . L . BARRINGER and R. D. BUIqAG
A O) ct-
-r
90
60
30
II) 0
tO n" ,,l.a
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_~. . . .
i ....
~i---
. . . . ~-
-30
t~
"l-
-60
i
-90
B (~ ttO
i
~
i
L
t
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I
I
I
I
I
90
60
3o
•~
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u ....
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-30 i--II
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-6o
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COMBINED BLOCKADE
i -90
- -
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30
i
20
J
i
10
1
z
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Blood Pressure
i
-10
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Change
Fig. 3. Reflex heart rate changes (bpm) on the ordinate plotted against corresponding changes m mean pressure (mm Hg) on the abscissa before and after combined autonomic blockade with propranolol and atropine in 4-month (upper panel A) and 14-month (lower panel B)-old rats. Data expressed as averages + SEM.
Reflex bradycardia reduced more in 14-month old rats by either ~-adrenergic or cholinergic blockade All reflex heart rate responses were quantitatively reduced by either [3-adrenergic (Table 3) or cholinergic blockade (Table 4) as compared with those recorded in the same rats prior to blockade (Table 1). To determine whether these reductions occurred equally in both age groups, an analysis of covariance was performed using heart rate responses prior to blockade as
71
AGE-RELATED BAROREFLEX A'I~ENUATION
TABLE 2 . EFFECTS OF AUTONOMIC BLOCKADE ON BASELINES FOR MEAN PRESSURE AND HEART RATE IN CONSCIOUS 4-MONTH AND 14-MONTH OLD RATS
Age Group
Type of Autonomic Blockade Cholinergic ~5-adrenergic
Without
Combined
(A) Mean Pressure (ram Hg) 4-month 14-month
100 ± 108 ±
1 3
102 --110 ±
3 4
105 - 5 109 ± 3
98 ± 11 111 ± 5
311 ± 9 a 295 +-- 3 ~
336 ± 305 ±
(B) Heart Rate (bpm) 4-month 14-month
362 - 10 351 ± 7
417 --- 10 a 389 ± 11 a
8a 3a
~p < 0.005 as compared using a paired t-test with corresponding average from the same rats without blockade.
covariants. Responses after blockade were then expressed not only as absolute values, but also as adjusted means. For reflex tachycardia, adjusted means obtained after [3-adrenergic (Table 3B) or cholinergic (Table 4B) blockade in 14-month-old rats appeared to be smaller, but were not significantly different between age groups. In contrast, for reflex bradycardia, adjusted means after blockade with either drug were consistently smaller in 14-month-old rats. Bradycardia caused by blood pressure increases of 25 mm Hg or more after 13-adrenergic blockade (Table 3A) and of 20 mm Hg or more after cholinergic blockade (Table 4A) was significantly smaller in 14-month-old rats than in younger rats. Since blockade with either atropine or propranolol reduced reflex TABLE 3. REFLEX HEART RATE RESPONSES (bpm) ELICITED IN CONSCIOUS 4-MONTH AND 14-MONTH-OLD RATS FOLLOWING B-ADRENERGIC BLOCKADE
Age Group
5
10
Change in Mean Pressure (ram Hg) 15 20
25
30
(A) Reflex Bradycardia to Phenylephrine 4-month
-4
± 5
-
14-month
10.53
-2 -
-7 -
± 1 9.27
--- 2 10.92
- 5 --- 1 - 9.48
-11 -
± 2
11.09
-8 ± 2 - 10.41
-18 -
± 3
14.91
-11 - 2 - 11.42
-25 -
± 4
17.99
-32
+ 5
- 22.27
- 1 4 +__ 2 - 12.99 a
-16 ± 3 - 13.79 a
24 ± 4 17.74 13 ± 2
27 -+ 4 18.51 13 ± 3
F-ratio = 2.40, p < 0.04
(B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
3 ± 1 8.39 3 ± 1 8.88
7 -- 1 10.75 5 --- 2 9.55
11 -+ 2 11.24 7 ± 2 9.77
18 ± 4 14.87 11 ± 2 11.57
11.04
10.94
F-ratio = 1.32, p > 0.2 *Averages ± SEM with bold numbers below each average representing adjusted means obtained by covariant analysis. ap < 0.05 as compared using N e w m a n - K e u l s ' multiple range test with corresponding average of 4-monthold rats.
72
D. L BARRINGERand R. D. BU/qAG TABLE 4. REFLEX HEART RATE RESPONSES(bpm) ELICITED IN CONSCIOUS4-MONTH AND 14-MONTH-OLDRATS FOLLOWINGCHOLINERGICBLOCKADE*
Age Group
5
10
Change in Mean Pressure (mm Hg) 15 20
25
30
- 3 0 -+ 3
-35 _+ 4
(A ) Reflex Bradycardia to Phenylephrine 4-month
- 4 -- 1 - 5.15
14-month
- 9 +-- 1 - 9.15
- 4 _+ 1 -4.66
- 7 _+ 1 - 7.94
- 1 4 -- 2 -
14.22
-9 + 2 - 9.58
-21 _+ 3 - 20.33
- 28.32
-15 +- 3 -
14.56
a
- 33.85
-18 _+ 3 -
17.30
a
-21 _+ 3 - 20.56
a
F-ratio = 3.89, p < 0.003 (B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
8 -+ 2 15.91 3 -+ 1
14 -+ 3 18.70 8 -+ 2
24 -+ 4 23.35 14 --- 3
33 +- 5 27.70 19 -+ 4
42 -+ 5 32.95 25 -+ 4
52 -+ 6 39.09 28 -+ 4
12.84
15.40
20.40
20.40
23.79
25.42
F-ratio = 2. t0, p >0.06 *Averages _.+ S E M with bold numbers b e l o w each average representing adjusted means obtained by covariant analysis. ap < 0 . 0 5 as c o m p a r e d using N e w m a n - K e u l s ' multiple range test with corresponding average of 4 - m o n t h - o l d rats.
bradycardia m o r e in 14-month-old rats than in y o u n g e r rats, both sympathetic and parasympathetic contributions to reflex decreases in heart rate must have been diminished by age.
Abolition o f reflex heart rate responses following combined blockade W h e n drug infusions were repeated f o l l o w i n g pretreatment with both atropine and propranolol, arterial pressure still rose or fell as phenylephrine or s o d i u m nitroprusside was infused, but reflex heart rate changes were no longer elicited (Figure 3).
DISCUSSION U p o n c o m p a r i n g 4 - m o n t h and 14-month-old f e m a l e S p r a g u e - D a w l e y rats we found the following. First, reflex heart rate responses to infusions o f phenylephrine or sodium nitroprusside were consistently w e a k e r in 14-month-old rats than in y o u n g e r rats (Table 1). Second, regardless o f the type of p h a r m a c o l o g i c blockade, baseline heart rates were altered similarly in all rats thereby indicating that basal sympathetic and parasympathetic tone was unaffected by age (Table 2). Third, selective blockade with either atropine or propranolol reduced reflex tachycardia equally, but the reduction in reflex bradycardia was greater in 14-month-old rats than in y o u n g e r rats. These findings suggest that chronotropic baroreflexes in rats b e c o m e impaired with age, and this i m p a i r m e n t is due to selective reduction in a u t o n o m i c control o f reflex bradycardia. Baroreflex i m p a i r m e n t in 14-month-old rats m a y reflect a sluggishness o f either baroreflex c o m p o n e n t s or the heart itself. H o w e v e r , although reflex heart rate responses were generally
AGE-RELATED BAROREFLEX ATTENUATION
73
weaker in 14-month old rats age-related differences were significant for pressure changes of 20 mm Hg or more not only prior to blockade (Table 1), but also after 13-adrenergic (Table 3) or cholinergic (Table 4) blockade. If this means that the heart in old rats still responded normally to pressures below 15 mm Hg, either before or after autonomic blockade, then reduced responsiveness to higher pressures would more likely be due to baroreflex saturation, rather than to cardiac sluggishness. Abolition of all heart rate responses by combined blockade with atropine and propranolol (Figure 3) indicates that they were elicited reflexly through efferent parasympathetic and sympathetic pathways (Bufiag et al., 1975; Coleman, 1980; Glick and Braunwald, 1965). After parasympathetic blockade with atropine, only sympathetic activity would remain to increase during reflex tachycardia and decrease during reflex bradycardia. Conversely, after 13adrenergic blockade with propranolol, unopposed vagal or parasympathetic activity would increase during reflex bradycardia and decrease during reflex tachycardia. Therefore, reflex responsiveness remaining after cholinergic blockade would reflect sympathetic activity, whereas responsiveness remaining after [3-adrenergic blockade would reflect parasympathetic activity. Hence, while parasympathetic and sympathetic contributions to reflex tachycardia were apparently equal in both age groups (Tables 3B and 4B), both autonomic contributions during reflex bradycardia must have been weaker in 14-month-old rats because their responses after either 13-adrenergic (Table 3A) or cholinergic (Table 4A) blockade were smaller than those in younger rats. Although Rothbaum et al. (1974) did not study reflex tachycardia, they concluded that only the parasympathetic contribution to reflex bradycardia diminishes with age. The difference between this conclusion and ours could be partly due to their use of older (i.e., 12- and 24-month-old) male Wistar rats, and autonomic evaluation only of reflex bradycardia at sustained 55 mm Hg increases in systolic pressure. Instead, we studied younger (i.e., 4- and 14-month-old) female Sprague-Dawley rats, and examined both increases and decreases in arterial pressure that occurred gradually. Because vagal tone changes earlier and more rapidly than sympathetic tone (Wang and Borison, 1947), the abrupt pressure changes they used could have altered vagal tone alone without affecting sympathetic tone. By contrast, with the more gradual changes in pressure used here, there would have been more time for changes in sympathetic tone so that both sympathetic and parasympathetic contributions were being assessed (Coleman, 1980). Reflex bradycardia in conscious male Sprague-Dawley rats was attributed mainly to parasympathetic activation by Stornetta et al. (14) because they found it abolished following cholinergic blockade with methylatropine but unaffected following 13-adrenergic blockade with atenolol. Apart from differences in the antagonist drugs used, the Sprague-Dawley rats they studied differ from ours not only in sex (i.e., theirs were males while ours were females) but probably also in age (i.e., with a body weight range of 300 to 350 g their male rats were about 2-3 months old). Accordingly, the differences between their results and ours could be due to differences in antagonist drugs and rat ages, and also to sex differences since autonomic regulation in rats has been shown to be predominantly parasympathetic in males and sympathetic in females (Bufiag et al., 1975). Age could affect various baroreflex arc functions like arterial wall distensibility, afferent nerve discharge pattern, central integration, efferent transmission, or myocardial responsiveness. Explanations based on reduced arterial wall stretching or afferent nerve discharge are unlikely because despite decreased aortic arch distensibility, baroreceptor afferent discharge
74
D. L. BARRINGER and R. D. BUNAG
rate remains unaltered with age (Andresen, 1984). This implies that despite the diminution in vessel wall. elasticity the baroreceptor sensitivity in older rats has increased and afferent discharge rates have remained constant. Reduced myocardial ~-adrenergic sensitivity is also unlikely because pithed Sprague-Dawley rats, regardless of age, have similar heart rate responses to isoproterenol (Docherty et al., 1986). Based on the information now available, the most probable explanation for baroreflex attenuation with age would be an altered central processing of baroreceptor afferent signals with resultant modification of chronotropic control. Thus, the sympathetic inhibition and bradycardia produced by both phenylephrine infusions and aortic nerve stimulation were weaker in 9-month than in 2-month-old rats (Tanabe and Bufiag, 1989). Furthermore, the parasympathetic (Table 3) and sympathetic (Table 4) contributions to reflex bradycardia that occurred here during phenylephrine infusions were reduced in 14month-old rats. Because both atropine and propranolol can cross the blood-brain barrier, it is conceivable that these antagonists may have acted centrally, at least in part, to bring about the age-related changes in reflex bradycardia. Nonetheless, our findings collectively suggest that the most likely explanation for baroreflex impairment with age would be an altered central integration of afferent or efferent impulses. Why impairment of autonomic mechanisms in 14-month-old female rats was limited only to bradycardia while tachycardia was unaltered is unknown. Autonomic regulation of heart rate normally consists of a delicate reciprocal balance between parasympathetic (i.e., cholinergic) deceleration and sympathetic (i.e., f3-adrenergic) acceleration. Baroreceptors in the carotid sinuses and aortic arch adjust this balance constantly by altering efferent parasympathetic and sympathetic neural activity to produce bradycardia or tachycardia as blood pressure rises or falls, and the predominant mechanism responsible for adjustment probably varies with the animal species. In man and in dogs, reflex changes are governed mainly by vagal or parasympathetic activity which increases during reflex bradycardia (Greene and Bachand, 1971 ; Higgins et al., 1973; Scher and Young, 1970) and conversely, decreases during reflex tachycardia (Pickering et al., 1972; Vatner et al., 1974). Instead of conforming with this scheme, however, rats have marked sexual predispositions that incline males to parasympathetic predominance and females to sympathetic predominance (Bufiag et al., 1975). As aging tends to tilt the autonomic balance further towards sympathetic predominance (Bufiag et al., 1990) the progressive enhancement in sympathetic activity would be accompanied by a reciprocal diminution in parasympathetic activity which may have started already in the 14-month-old females studied here. But even if we assume that the normal autonomic balance deteriorates gradually with age, it still remains unclear exactly how reflex bradycardia alone could be selectively impaired since there are no baroreflex centers or pathways that mediate either bradycardia or tachycardia exclusively. In summary, we have shown that baroreflex sensitivity was less in 14-month-old rats than in 4-month-old rats. Reflex chronotropic responses were reduced further following cholinergic blockade with atropine or 13-adrenergic blockade with propranolol. Since drug-induced inhibition was greater in 14-month-old rats during reflex bradycardia, but similar in both groups during reflex tachycardia, we conclude that autonomic regulation, consisting of concurrent parasympathetic activation and sympathetic withdrawal during reflex bradycardia, was also reduced by age. Acknowledgments -- This work was supportedby researchgrants HL 37980 and HL 39383 from the NationalHeart,
Lung and Blood Institute.
AGE-RELATEDBAROREFLEXATTENUATION
75
REFERENCES ANDRESEN,M.C. Short- and long-term determinants of baroreceptor function in aged normotensive and spontaneously hypertensive rats. Circ. Res. 54, 750-759, 1984.
BADER,H. Dependence of wall stress in the human thoracic aorta on age and pressure. Circ. Res. 20, 354-361, 1967. BARR1NGER,D.L. and BU~AG,R.D. Uneven blunting of chronotropic baroreflexes in obese Zucker rats. Am. J. Physiol. 256, H417-H421, 1989. BERKOW1TZ,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, 447-455, 1969. BMDP STATISTICALSOFTWARE.BMDP Statistical Software Inc., 1964 Westwood Blvd. Suite 202, Los Angeles, CA 90025, 1985. BRUNING,J.L. and KINTZ, B.L. Computational Handbook of Statistics, Glenview, IL; Scott, Foresman and Company, 3rd Edition, 1987. BUlqAG,R.D. and MIYAJIMA,E. Baroreflex impairment precedes hypertension during chronic cerebroventricular infusion of hypertonic sodium chloride in rats. J. Clin. Invest. 74, 2065-2073, 1984. BUSAG, R.D., TERAVAINEN,T., and ERIKSSON,L. Enhanced sympathetic mediation of chronotropic baroreflexes in old Sprague-Dawley rats. Mech. Ageing Dev. 53, 195-208, 1990. BUlqAG,R.D., WALASZEK,E.J., and MUET1NG,N. Sex differences in reflex tachycardia induced by hypotensive drugs in unanesthetized rats. Am. J. Physiol. 229,652~556, 1975. COLEMAN,T.G. Arterial baroreflex control of heart rate in the conscious rat. Am. J. Physiol. 238, H515-H520, 1980. DOCHERTY,J.R., FITZGERALD,D., and O'MALLEY,K. Age-related reduction of baroreflex tachycardia without loss of 13-adrenoceptor-mediatedtachycardia in Sprague-Dawley rats. J. Cardiovasc. Pharmacol. 8, 376-380, 1986. FERRARI, A.U., DAFFONCHIO,A,, ALBERGATH,F., and MANCIA,G. Inverse relationship between heart rate and blood pressure variabilities in rats. Hypertension 10, 533-537, 1987. GLICK, G. and BRAUNWALD,E. Relative roles of the sympathetic and parasympathetic nervous systems in the reflex control of heart rate. Circ. Res. 14, 363-375, 1965. GREENE,N.M. and BACHAND,R.G. Vagal component of the chronotropic response to baroreceptor stimulation in man. Am. HeartH. 82, 22-27, 1971. Hlo31NS, C.B., VATNER,S.F., and BRAUNWALD,E. Parasympathetic control of the heart. Pharmacol. Rev. 25, 119-155, 1973. Hi~"rzE, J.L., ED. Number Cruncher Statistical System, (Dr. Jerry L. Hintze, Kaysville, UT), 21.1-21.8, 1986. lhcrmR1NG,T.G., GRIBBIN,B., PETERSEN,E.S., CUNNINGHAM,D., and SLEIGHT,P. Effects of autonomic blockade on the baroreflex in man at rest and during exercise. Circ. Res. 30, 177-185, 1972. ROTHBAUM,D.A., SHAW,D.J., ANGELL,C.S., and SHOCK,N.W. Age differences in the baroreceptor response of rats. J. Geront. 29, 488-492, 1974. SCHER,A.M. and YOUNG,A.C. Reflex control of heart rate in the unanesthetized dog. Am. J. Physiol. 218, 780-789, 1970. STORNETTA,R.L., GUYENET, P.G., and McCARTY, R.C. Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats. J. Auton. Nerv. Syst. 20, 121-127, 1987. TANABE,S. and BURAG,R.D. Age-related central and baroreceptor impairment in female Sprague-Dawley rats. Am. J. Physiol. 256, H1399-H1406, 1989. VATNER, S.F., HIGGINS,C . l . , and BRAUNWALD,E. Sympathetic and parasympathetic components of reflex tachycardia induced by hypotension in conscious dogs with and without heart failure. Cardiovasc. Res. 8, 153-161, 1974. WANG, S.C. and BORISON,H.L. An analysis of the carotid sinus cardiovascular reflex mechanism. Am. J. Physiol. 150, 712-21, 1947.
0531-5565/91 $3.00 + .00 Copyright©1991 PergamonPress plc
AUTONOMIC REGULATION OF REFLEX BRADYCARDIA IN RATS DECLINES WITH AGE
DONNA L. BARRINGERand RUBEN D. BU/qAG Department of Pharmacology, College of Health Sciences and Hospital, Universityof Kansas Medical Center, Kansas City, KS 66103
A b s t r a c t - Heart rate responses, elicited reflexly by elevating blood pressure with phenylephrine or lowering it with sodium nitroprusside, were compared in groups of rats aged 4 and 14 months. All tests were done while the rats were awake to avoid artefacts due to anesthesia. Parasympathetic and sympathetic contributions were assessed by repeating baroreflex tests after cholinergic blockade with atropine or 13-adrenergic blockade with propranolol. Magnitude of reflex bradycardia and tachycardia was initially smaller in the 14-month-old rats than in the younger rats thereby indicating that baroreflex sensitivity had diminished with age. After 13-adrenergic or cholinergic blockade, adjusted means (obtained by covariance analysis) for reflex tachycardia did not differ significantly between rat groups, but those for reflex bradycardia were significantly smaller in the 14-month-old rats than in the younger rats. Selective attenuation of reflex bradycardia after either cholinergic or 13adrenergic blockade indicates that concurrent parasympathetic activation and sympathetic withdrawal during reflex bradycardia were also reduced in 14-month-old rats. These results suggest that as autonomic mediation of reflex bradycardia diminishes with age, old rats may no longer be able to slow the heart as easily whenever blood pressure rises, but they can still accelerate it whenever blood pressure falls. Key Words: aging, baroreflexes, ~-adrenergic blockade, blood pressure, conscious rats, cholinergic blockade, heart rate, parasympathetic, sympathetic
INTRODUCTION BAROREFLEX ATI'ENUATIONin old rats has been described previously (Docherty et al., 1986; Rothbaum et al., 1974; Tanabe and Bufiag, 1989), but underlying autonomic mechanisms remain uncertain. Of the studies that have used selective pharmacological blockade to identify sympathetic and parasympathetic contributions to heart rate reflexes in dogs (Berkowitz et al., 1969; Glick and Braunwald, 1965; Scher and Young, 1970; Vatner et al., 1974) and rats (Ferrari et al. 1987; Rothbaum et al., 1974; Stornetta et al., 1987), only one has described an
Correspondence to: Ruben D. Bufiag, Departmentof Pharmacology,Universityof Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS 66103. (Received 22 March 1990; Accepted 1 October 1990) 65
66
D. L. BARRINGER and R. D. BU/qAG
age-related attenuation of vagal tone during reflex bradycardia (Rothbaum et al., 1974) and possible changes in attendant sympathetic mediation were not explored. In anesthetized 9-month rats, Tanabe and Bufiag (1989) recently showed that while baroreflex sensitivity was still adequate at normotensive pressures, it was already impaired at pressures above 130 mm Hg. Whether a similar age-related baroreflex impairment would be demonstrable in conscious rats is unknown. Accordingly, in the present studies we compared reflex heart rate responses in conscious 4-month and 14-month-old rats to determine whether sympathetic and parasympathetic contributions to heart rate control change with age. Reflex heart rate responses were elicited by elevating blood pressure with phenylephrine or lowering it with sodium nitroprusside. Cholinergic and [3-adrenergic contributions were later quantified by using atropine and propranolol to block efferent parasympathetic and sympathetic pathways, respectively. MATERIALS AND METHODS Ten 4-month-old and thirteen 14-month-old female Sprague-Dawley rats were purchased from SASCO Inc (Omaha, NE). All rats were transiently anesthetized for surgical implantation of indwelling vascular cannulas. One week later, heart rates were recorded as blood pressure was altered by infusing phenylephrine or sodium nitroprusside intravenously while the rats were awake. For baroreflex testing, drugs were infused five times on postoperative days 7 and 8. On both days, baroreflex tests were first done prior to antagonist pretreatment and then repeated following cholinergic or 13-adrenergic blockade. Additionally, on day 8, a third test was done following combined cholinergic and 13-adrenergic blockade.
Surgical preparation for baroreflex testing After anesthetizing each rat by intraperitoneal injection of a mixture of ketamine (10 mg/ 100 g as a dissociative anesthetic) and acepromazine (0.1 mg/100 g as a supplementary tranquilizer), indwelling cannulas were inserted separately into the right femoral artery for recording blood pressure and the ipsilateral vein for infusing drugs (Barringer and Bufiag, 1989; Bufiag and Miyajima, 1984). Cannulas consisted of two pieces of polyethylene tubing (a 2-4 cm length of PE-10 threaded and heat-fused into a 13-18 cm length of PE-50 tubing) with a total dead-space volume of 0.05-0.1 ml. The end with PE-10 tubing was inserted directly into the blood vessel while the larger bore PE-50 tubing was passed subcutaneously to emerge at the nape. To keep cannulas patent, immediately after surgical implantation and on the third and fifth postoperative days, they were cleared by rapidly injecting 0.5 ml saline, then filled with heparinized (30 U/ml) saline. Following surgery and between recording sessions, rats were kept in individual cages in an air-conditioned room with unrestricted access to food (Rodent Laboratory Chow, Ralston Purina Company, St. Louis, MO) and water.
Recording reflex chronotropic responses to intravenously-infused drugs One week after surgical cannulation, with each awake rat kept in its own open-topped plastic cage, pulsatile pressure was recorded on a Gould polygraph by connecting the arterial cannula through PE-50 tubing to a small-volume-displacement pressure transducer (PI0EZ from Spectramed Inc, Critical Care Division, Oxnard, CA) placed at the same level as the rat. Heart
AGE-RELATED BAROREFLEX A'I'rENUATION
67
rate was monitored simultaneously by triggering a biotachometer with the phasic pressure signal from the transducer. The venous cannula was connected to a syringe pump (Harvard Apparatus, South Natick, MA) for drug infusions. After connecting both cannulas, 45--60 min was allowed to lapse without further manipulation so that the rat could become accustomed to the recording environment. Arterial pressures and heart rates were then recorded continuously during slow intravenous infusions, first of phenylephrine and then of sodium nitroprusside. Doses (nanograms salt/100 grams body weight/min) ranging from 750 to 1500 for L-phenylephrine hydrochloride and 550 to 1100 for sodium nitroprusside were infused at a rate of 20 Ixl/min for 30 seconds using a syringe pump. All infusions were regulated to alter arterial pressure progressively by 30-40 mm Hg within 30 seconds. Phenylephrine was diluted with isotonic saline while sodium nitroprusside was prepared in 5% dextrose and protected from light to retard spontaneous decay. Reflex heart rate responses to drug-induced changes in arterial pressure were recorded five times on two successive days. On the seventh day after cannulation, a control recording was taken first, and then followed by a second recording 15 min after either cholinergic blockade with atropine sulfate, or B-blockade with propranolol hydrochloride; doses of 0.1 mg/100 g IV used for both drugs have previously been found effective in abolishing all heart rate responses elicited reflexly by phenylephrine or sodium nitroprusside (Barringer and Bufiag, 1989; Coleman, 1980). On the eighth day, baroreflex responses were recorded three more times: (1) an initial test before antagonist pretreatment, (2) a second test 15 min after blockade with either atropine or propranolol (using the antagonist that had not been used the previous day), and (3) a third test 15 min after combined blockade induced by added injection of the second antagonist.
Data analysis and statistics All data (except adjusted means) were expressed as averages + SEM. Mean arterial pressure was calculated as diastolic pressure plus one-third of the pulse pressure. Baroreflex responses during infusions of phenylephrine or sodium nitroprusside were analyzed by reading heart rate values at each 5 mm Hg step-wise change in mean arterial pressure (up to 30 mm Hg). Differences in reflex heart rate changes between young and old groups were assessed using a repeated measures analysis of variance. Mainly because initial reflex respoffses differed between age groups, and also because baseline heart rates were altered significantly following treatment with atropine or propranolol, reflex responsiveness following antagonist treatment was assessed by covariance analysis using data obtained after blockade as the dependent variable and data recorded from the same rats prior to blockade as the covariate. Whenever F-Ratios were significant, Newman-Keuls' multiple range test (Bruning and Kintz, 1987) was used to determine significance between pairs of means. P-values of 0.05 or less were considered statistically significant. Repeated measures analysis of covariance was done on an IBM Model 4381 using BMDP Statistical Software (BMDP, 1985), while all other statistical tests were done on an IBM XT computer with the Number Cruncher Statistical System (Hintze, 1986). RESULTS
Stability of reflex responsiveness in conscious rats In all rats, intravenous infusions of phenylephrine gradually elevated mean arterial pressure and reflexly slowed heart rate (Figure 1) while similar infusions of sodium nitroprusside had
68
D. L. BARRINGER and R D. BUIqAG
A
B
200-~
Fig. I. Changes in pulsatile pressure (mm Hg, lower panel) and heart rate (bpm, upper panel) during continuous phenylephrine infusion in a 4-month-old (A) and 14-month-old(B) rat. Chart speed 125 mm/min. Arrow indicates beginning of infusion. opposite effects of gradually lowering mean arterial pressure and reflexly increasing heart rate (Figure 2). Blood pressure rose or fell by 5 - 8 mm Hg per 5 seconds and with either drug, magnitude of the reflex heart rate response increased as the corresponding change in mean pressure became greater. Drug doses required to lower or elevate blood pressure by 30 to 40 mm Hg were similar regardless of age. Within each rat group, initial chronotropic responses to increases or decreases in blood pressure were qualitatively and quantitatively the same on days 7 and 8. The absence of significant differences between days indicated that antagonists administered on the first day were no longer present on the second day so that effects of another antagonist could be tested. Since it also shows that reflex responsiveness was reproducible from day to day, results from both days were pooled (Table l) to obtain single values representing average reflex responses prior to antagonist treatment.
A
B
Fig. 2. Changes in pulsatile pressure (mm Hg, lower panel) and heart rate (bpm, upper panel) during continuous sodium nitroprusside infusion in a 4-month-old (A) and 14-month-old(B) rat. Chart speed 125 mm/min. Arrow indicates beginning of infusion.
69
AGE-RELATED BAROREFLEX A'ITENUAT1ON TABLE 1. AVERAGE REFLEX HEART RATE RESPONSES ( b p m ) ELICITED BY INTRAVENOUSLYINFUSEDDRUGS IN CONSCIOUS 4-MONTH AND 14-MONTH OLD RATS*
Age Group
5
10
Change in Mean Pressure (Am Hg) 15 20
25
30
-51 -+ 4 -31 + 5a
- 5 9 -4- 5 - 3 5 --- 5a
66 --- 9 40 + 5a
79 --- 10 45 -+ 7a
(A) Reflex Bradycardia to Phenylephrine 4-month 14-month
- 1 0 -- 2 - 6 --- 2
- 1 9 --- 2 - 1 3 -4- 2
- 3 2 --- 5 -21 +- 4a
- 4 2 --- 5 - 2 7 + 4a
F-ratio = 10.75, p < 0.004
(B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
9 +-- 3 3 --- 1
21 --- 4 12 +-- 2
35 +-- 5 23 --- 3
48 --- 6 32 -+- 4 a
F-ratio = 10.45, p < 0.004 *Expressed as averages ___ SEM from 10 rats in each group by combining data on days 7 and 8. ap < 0.05 as compared with corresponding average for 4-month-old rats using Newman-Keuls' multiple range test.
Baroreflex attenuation in 14-month old rats M a g n i t u d e o f either reflex bradycardia or tachycardia was generally smaller in 14-month than in 4 - m o n t h - o l d rats. F o r reflex bradycardia p r o d u c e d during p h e n y l e p h r i n e - i n d u c e d pressor responses o f 15 m m Hg or m o r e , or for reflex tachycardia p r o d u c e d during s o d i u m nitroprusside induced depressor responses o f - 2 0 m m H g or m o r e (Table 1) the differences b e t w e e n rat groups w e r e significant. By indicating that baroreflex sensitivity, whether manifested as bradycardia or tachycardia, was attenuated in the older rats this finding confirms the age-related baroreflex i m p a i r m e n t reported p r e v i o u s l y by T a n a b e and Bufiag (1989). B e c a u s e heart rate reflexes n o r m a l l y d e p e n d on efferent parasympathetic and sympathetic pathways, it was considered possible that reduced baroreflex sensitivity in old rats m a y reflect an a u t o n o m i c imbalance. This possibility was tested by recording reflex responses in the same rats after appropriate p h a r m a c o l o g i c a l blockade: [3-adrenergic blockade with propranolol to reveal parasympathetic m e c h a n i s m s , or cholinergic blockade with atropine to reveal sympathetic m e c h a n i s m s .
Selective autonomic blockade altered basal heart rate but not blood pressure Initial baselines for either m e a n pressure or heart rate did not differ significantly b e t w e e n age groups (Table 2). Basal heart rates w e r e elevated f o l l o w i n g cholinergic b l o c k a d e with atropine and l o w e r e d f o l l o w i n g [3-adrenergic b l o c k a d e with propranolol, while those for m e a n pressure w e r e unaffected (Table 2). D i f f e r e n c e s b e t w e e n age groups in ensuing heart rate changes w e r e not significant; cholinergic b l o c k a d e increased baseline heart rates (bpm) by 55 +_ 11 in 4 - m o n t h - o l d rats and 36 ___ 8 in 14-month-old rats, while [3-adrenergic blockade decreased baseline heart rates by 51 --- 6 and 56 --- 8, respectively. Regardless o f age, heart rates f o l l o w i n g c o m b i n e d b l o c k a d e with both atropine and propranolol w e r e significantly l o w e r than the original basal levels (Table 2).
70
D . L . BARRINGER and R. D. BUIqAG
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-r
90
60
30
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i ....
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. . . . ~-
-30
t~
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-60
i
-90
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i
~
i
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t
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I
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I
I
I
90
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u ....
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i -90
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i
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J
i
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z
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i
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Fig. 3. Reflex heart rate changes (bpm) on the ordinate plotted against corresponding changes m mean pressure (mm Hg) on the abscissa before and after combined autonomic blockade with propranolol and atropine in 4-month (upper panel A) and 14-month (lower panel B)-old rats. Data expressed as averages + SEM.
Reflex bradycardia reduced more in 14-month old rats by either ~-adrenergic or cholinergic blockade All reflex heart rate responses were quantitatively reduced by either [3-adrenergic (Table 3) or cholinergic blockade (Table 4) as compared with those recorded in the same rats prior to blockade (Table 1). To determine whether these reductions occurred equally in both age groups, an analysis of covariance was performed using heart rate responses prior to blockade as
71
AGE-RELATED BAROREFLEX A'I~ENUATION
TABLE 2 . EFFECTS OF AUTONOMIC BLOCKADE ON BASELINES FOR MEAN PRESSURE AND HEART RATE IN CONSCIOUS 4-MONTH AND 14-MONTH OLD RATS
Age Group
Type of Autonomic Blockade Cholinergic ~5-adrenergic
Without
Combined
(A) Mean Pressure (ram Hg) 4-month 14-month
100 ± 108 ±
1 3
102 --110 ±
3 4
105 - 5 109 ± 3
98 ± 11 111 ± 5
311 ± 9 a 295 +-- 3 ~
336 ± 305 ±
(B) Heart Rate (bpm) 4-month 14-month
362 - 10 351 ± 7
417 --- 10 a 389 ± 11 a
8a 3a
~p < 0.005 as compared using a paired t-test with corresponding average from the same rats without blockade.
covariants. Responses after blockade were then expressed not only as absolute values, but also as adjusted means. For reflex tachycardia, adjusted means obtained after [3-adrenergic (Table 3B) or cholinergic (Table 4B) blockade in 14-month-old rats appeared to be smaller, but were not significantly different between age groups. In contrast, for reflex bradycardia, adjusted means after blockade with either drug were consistently smaller in 14-month-old rats. Bradycardia caused by blood pressure increases of 25 mm Hg or more after 13-adrenergic blockade (Table 3A) and of 20 mm Hg or more after cholinergic blockade (Table 4A) was significantly smaller in 14-month-old rats than in younger rats. Since blockade with either atropine or propranolol reduced reflex TABLE 3. REFLEX HEART RATE RESPONSES (bpm) ELICITED IN CONSCIOUS 4-MONTH AND 14-MONTH-OLD RATS FOLLOWING B-ADRENERGIC BLOCKADE
Age Group
5
10
Change in Mean Pressure (ram Hg) 15 20
25
30
(A) Reflex Bradycardia to Phenylephrine 4-month
-4
± 5
-
14-month
10.53
-2 -
-7 -
± 1 9.27
--- 2 10.92
- 5 --- 1 - 9.48
-11 -
± 2
11.09
-8 ± 2 - 10.41
-18 -
± 3
14.91
-11 - 2 - 11.42
-25 -
± 4
17.99
-32
+ 5
- 22.27
- 1 4 +__ 2 - 12.99 a
-16 ± 3 - 13.79 a
24 ± 4 17.74 13 ± 2
27 -+ 4 18.51 13 ± 3
F-ratio = 2.40, p < 0.04
(B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
3 ± 1 8.39 3 ± 1 8.88
7 -- 1 10.75 5 --- 2 9.55
11 -+ 2 11.24 7 ± 2 9.77
18 ± 4 14.87 11 ± 2 11.57
11.04
10.94
F-ratio = 1.32, p > 0.2 *Averages ± SEM with bold numbers below each average representing adjusted means obtained by covariant analysis. ap < 0.05 as compared using N e w m a n - K e u l s ' multiple range test with corresponding average of 4-monthold rats.
72
D. L BARRINGERand R. D. BU/qAG TABLE 4. REFLEX HEART RATE RESPONSES(bpm) ELICITED IN CONSCIOUS4-MONTH AND 14-MONTH-OLDRATS FOLLOWINGCHOLINERGICBLOCKADE*
Age Group
5
10
Change in Mean Pressure (mm Hg) 15 20
25
30
- 3 0 -+ 3
-35 _+ 4
(A ) Reflex Bradycardia to Phenylephrine 4-month
- 4 -- 1 - 5.15
14-month
- 9 +-- 1 - 9.15
- 4 _+ 1 -4.66
- 7 _+ 1 - 7.94
- 1 4 -- 2 -
14.22
-9 + 2 - 9.58
-21 _+ 3 - 20.33
- 28.32
-15 +- 3 -
14.56
a
- 33.85
-18 _+ 3 -
17.30
a
-21 _+ 3 - 20.56
a
F-ratio = 3.89, p < 0.003 (B) Reflex Tachycardia to Sodium Nitroprusside 4-month 14-month
8 -+ 2 15.91 3 -+ 1
14 -+ 3 18.70 8 -+ 2
24 -+ 4 23.35 14 --- 3
33 +- 5 27.70 19 -+ 4
42 -+ 5 32.95 25 -+ 4
52 -+ 6 39.09 28 -+ 4
12.84
15.40
20.40
20.40
23.79
25.42
F-ratio = 2. t0, p >0.06 *Averages _.+ S E M with bold numbers b e l o w each average representing adjusted means obtained by covariant analysis. ap < 0 . 0 5 as c o m p a r e d using N e w m a n - K e u l s ' multiple range test with corresponding average of 4 - m o n t h - o l d rats.
bradycardia m o r e in 14-month-old rats than in y o u n g e r rats, both sympathetic and parasympathetic contributions to reflex decreases in heart rate must have been diminished by age.
Abolition o f reflex heart rate responses following combined blockade W h e n drug infusions were repeated f o l l o w i n g pretreatment with both atropine and propranolol, arterial pressure still rose or fell as phenylephrine or s o d i u m nitroprusside was infused, but reflex heart rate changes were no longer elicited (Figure 3).
DISCUSSION U p o n c o m p a r i n g 4 - m o n t h and 14-month-old f e m a l e S p r a g u e - D a w l e y rats we found the following. First, reflex heart rate responses to infusions o f phenylephrine or sodium nitroprusside were consistently w e a k e r in 14-month-old rats than in y o u n g e r rats (Table 1). Second, regardless o f the type of p h a r m a c o l o g i c blockade, baseline heart rates were altered similarly in all rats thereby indicating that basal sympathetic and parasympathetic tone was unaffected by age (Table 2). Third, selective blockade with either atropine or propranolol reduced reflex tachycardia equally, but the reduction in reflex bradycardia was greater in 14-month-old rats than in y o u n g e r rats. These findings suggest that chronotropic baroreflexes in rats b e c o m e impaired with age, and this i m p a i r m e n t is due to selective reduction in a u t o n o m i c control o f reflex bradycardia. Baroreflex i m p a i r m e n t in 14-month-old rats m a y reflect a sluggishness o f either baroreflex c o m p o n e n t s or the heart itself. H o w e v e r , although reflex heart rate responses were generally
AGE-RELATED BAROREFLEX ATTENUATION
73
weaker in 14-month old rats age-related differences were significant for pressure changes of 20 mm Hg or more not only prior to blockade (Table 1), but also after 13-adrenergic (Table 3) or cholinergic (Table 4) blockade. If this means that the heart in old rats still responded normally to pressures below 15 mm Hg, either before or after autonomic blockade, then reduced responsiveness to higher pressures would more likely be due to baroreflex saturation, rather than to cardiac sluggishness. Abolition of all heart rate responses by combined blockade with atropine and propranolol (Figure 3) indicates that they were elicited reflexly through efferent parasympathetic and sympathetic pathways (Bufiag et al., 1975; Coleman, 1980; Glick and Braunwald, 1965). After parasympathetic blockade with atropine, only sympathetic activity would remain to increase during reflex tachycardia and decrease during reflex bradycardia. Conversely, after 13adrenergic blockade with propranolol, unopposed vagal or parasympathetic activity would increase during reflex bradycardia and decrease during reflex tachycardia. Therefore, reflex responsiveness remaining after cholinergic blockade would reflect sympathetic activity, whereas responsiveness remaining after [3-adrenergic blockade would reflect parasympathetic activity. Hence, while parasympathetic and sympathetic contributions to reflex tachycardia were apparently equal in both age groups (Tables 3B and 4B), both autonomic contributions during reflex bradycardia must have been weaker in 14-month-old rats because their responses after either 13-adrenergic (Table 3A) or cholinergic (Table 4A) blockade were smaller than those in younger rats. Although Rothbaum et al. (1974) did not study reflex tachycardia, they concluded that only the parasympathetic contribution to reflex bradycardia diminishes with age. The difference between this conclusion and ours could be partly due to their use of older (i.e., 12- and 24-month-old) male Wistar rats, and autonomic evaluation only of reflex bradycardia at sustained 55 mm Hg increases in systolic pressure. Instead, we studied younger (i.e., 4- and 14-month-old) female Sprague-Dawley rats, and examined both increases and decreases in arterial pressure that occurred gradually. Because vagal tone changes earlier and more rapidly than sympathetic tone (Wang and Borison, 1947), the abrupt pressure changes they used could have altered vagal tone alone without affecting sympathetic tone. By contrast, with the more gradual changes in pressure used here, there would have been more time for changes in sympathetic tone so that both sympathetic and parasympathetic contributions were being assessed (Coleman, 1980). Reflex bradycardia in conscious male Sprague-Dawley rats was attributed mainly to parasympathetic activation by Stornetta et al. (14) because they found it abolished following cholinergic blockade with methylatropine but unaffected following 13-adrenergic blockade with atenolol. Apart from differences in the antagonist drugs used, the Sprague-Dawley rats they studied differ from ours not only in sex (i.e., theirs were males while ours were females) but probably also in age (i.e., with a body weight range of 300 to 350 g their male rats were about 2-3 months old). Accordingly, the differences between their results and ours could be due to differences in antagonist drugs and rat ages, and also to sex differences since autonomic regulation in rats has been shown to be predominantly parasympathetic in males and sympathetic in females (Bufiag et al., 1975). Age could affect various baroreflex arc functions like arterial wall distensibility, afferent nerve discharge pattern, central integration, efferent transmission, or myocardial responsiveness. Explanations based on reduced arterial wall stretching or afferent nerve discharge are unlikely because despite decreased aortic arch distensibility, baroreceptor afferent discharge
74
D. L. BARRINGER and R. D. BUNAG
rate remains unaltered with age (Andresen, 1984). This implies that despite the diminution in vessel wall. elasticity the baroreceptor sensitivity in older rats has increased and afferent discharge rates have remained constant. Reduced myocardial ~-adrenergic sensitivity is also unlikely because pithed Sprague-Dawley rats, regardless of age, have similar heart rate responses to isoproterenol (Docherty et al., 1986). Based on the information now available, the most probable explanation for baroreflex attenuation with age would be an altered central processing of baroreceptor afferent signals with resultant modification of chronotropic control. Thus, the sympathetic inhibition and bradycardia produced by both phenylephrine infusions and aortic nerve stimulation were weaker in 9-month than in 2-month-old rats (Tanabe and Bufiag, 1989). Furthermore, the parasympathetic (Table 3) and sympathetic (Table 4) contributions to reflex bradycardia that occurred here during phenylephrine infusions were reduced in 14month-old rats. Because both atropine and propranolol can cross the blood-brain barrier, it is conceivable that these antagonists may have acted centrally, at least in part, to bring about the age-related changes in reflex bradycardia. Nonetheless, our findings collectively suggest that the most likely explanation for baroreflex impairment with age would be an altered central integration of afferent or efferent impulses. Why impairment of autonomic mechanisms in 14-month-old female rats was limited only to bradycardia while tachycardia was unaltered is unknown. Autonomic regulation of heart rate normally consists of a delicate reciprocal balance between parasympathetic (i.e., cholinergic) deceleration and sympathetic (i.e., f3-adrenergic) acceleration. Baroreceptors in the carotid sinuses and aortic arch adjust this balance constantly by altering efferent parasympathetic and sympathetic neural activity to produce bradycardia or tachycardia as blood pressure rises or falls, and the predominant mechanism responsible for adjustment probably varies with the animal species. In man and in dogs, reflex changes are governed mainly by vagal or parasympathetic activity which increases during reflex bradycardia (Greene and Bachand, 1971 ; Higgins et al., 1973; Scher and Young, 1970) and conversely, decreases during reflex tachycardia (Pickering et al., 1972; Vatner et al., 1974). Instead of conforming with this scheme, however, rats have marked sexual predispositions that incline males to parasympathetic predominance and females to sympathetic predominance (Bufiag et al., 1975). As aging tends to tilt the autonomic balance further towards sympathetic predominance (Bufiag et al., 1990) the progressive enhancement in sympathetic activity would be accompanied by a reciprocal diminution in parasympathetic activity which may have started already in the 14-month-old females studied here. But even if we assume that the normal autonomic balance deteriorates gradually with age, it still remains unclear exactly how reflex bradycardia alone could be selectively impaired since there are no baroreflex centers or pathways that mediate either bradycardia or tachycardia exclusively. In summary, we have shown that baroreflex sensitivity was less in 14-month-old rats than in 4-month-old rats. Reflex chronotropic responses were reduced further following cholinergic blockade with atropine or 13-adrenergic blockade with propranolol. Since drug-induced inhibition was greater in 14-month-old rats during reflex bradycardia, but similar in both groups during reflex tachycardia, we conclude that autonomic regulation, consisting of concurrent parasympathetic activation and sympathetic withdrawal during reflex bradycardia, was also reduced by age. Acknowledgments -- This work was supportedby researchgrants HL 37980 and HL 39383 from the NationalHeart,
Lung and Blood Institute.
AGE-RELATEDBAROREFLEXATTENUATION
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