Blockade of the angiotensin II (ANG II) type 1 receptor (AT1) has been shown to restore baroreflex sensitivity in rats and rabbits with experimental chronic heart failure (CHF). Because the modulation of baroreflex function in response to ANG II is mediated in part by AT1 receptors located in the area postrema, we hypothesized that lesions of the area postrema would prevent the enhancement in baroreflex function in response to AT1-receptor blockade in rabbits with pacing-induced CHF. Experiments were carried out on 24 male New Zealand White rabbits that were divided into sham (n = 12) and lesioned (n = 12) groups further divided into normal and CHF subgroups (n = 6 each). All rabbits were identically instrumented to measure cardiac external dimensions, central venous pressure, arterial pressure, heart rate (HR), and renal sympathetic nerve activity (RSNA). After 3–4 wk of pacing, baroreflex sensitivity (infusions of phenylephrine and nitroprusside) was evaluated before and after intravenous administration of the AT1-receptor antagonist L-158,809. Maximum baroreflex sensitivity in nonpaced rabbits was 5.4 ± 0.7 beats ⋅ min−1 ⋅ mmHg−1and 5.2 ± 0.5% of maximum/mmHg for HR and RSNA curves, respectively, and was not altered by L-158,809 in either intact or lesioned rabbits. In contrast, L-158,809 enhanced baroreflex sensitivity in intact rabbits with CHF (HR from 1.6 ± 0.3 to 4.1 ± 0.7 beats ⋅ min−1 ⋅ mmHg−1,P < 0.001; RSNA from 2.3 ± 0.2 to 4.9 ± 0.4% of maximum/mmHg,P < 0.001). However, in CHF rabbits with area postrema lesions, L-158,809 failed to enhance baroreflex sensitivity. Interestingly, area postrema lesions did not normalize the baroreflex in CHF rabbits. From these data we conclude that the area postrema mediates the normalization of baroreflex sensitivity after AT1 blockade in rabbits with CHF but does not modify resting baroreflex function.
- angiotensin II
- angiotensin receptors
- experimental heart failure
- central nervous system
- sympathetic nerve activity
the regulation of autonomic function in the chronic heart failure (CHF) state is significantly altered. Classically, sympathetic outflow is increased and vagal efferent outflow to the heart is decreased (13, 43, 46), resulting in increases in heart rate (HR) and total peripheral resistance in CHF. This sympathoexcitatory state contributes to the poor prognosis and high mortality rate of patients with severe CHF (38). Although the origin of the sympathoexcitation is not completely understood, it is known that ANG II, when elevated, can lead to augmentation of sympathetic outflow (39).
ANG II has been proposed to be an important neurotransmitter in various areas of the brain, and ANG II type 1 (AT1) receptors are ubiquitously distributed throughout those areas of the brain that are involved in the regulation of sympathetic outflow (5, 36). One area in which ANG II appears to play an important role in modulation of sympathetic outflow and baroreflex function is the area postrema (7, 9, 14). Because the area postrema is a circumventricular organ (CVO) devoid of a blood-brain barrier, AT1-receptor antagonists can easily block the effects of ANG II at this site. Clear evidence now exists for a neural pathway between the area postrema and the nucleus of the tractus solitarius (NTS) (35, 2), thereby further supporting the notion that the area postrema plays a modulatory role in baroreflex function and sympathetic outflow. Because this area of the brain stem appears to be pivotal for the role of ANG II in reducing baroreflex sensitivity (30), we chose to investigate the role of the area postrema in mediating the enhancement in baroreflex function in response to AT1 blockade in rabbits with pacing-induced CHF. We hypothesized that if central ANG II function was enhanced (either because of upregulation of receptors or because of increases in ANG II concentration) in the CHF state, the effects of AT1 blockade on baroreflex function would be abolished in CHF rabbits with lesions of the area postrema.
MATERIALS AND METHODS
Experiments were carried out on 24 male New Zealand White rabbits weighing between 2.5 and 3.5 kg. All surgical procedures and protocols were reviewed and approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Experiments were carried out under the guidelines for the care and use of experimental animals of the American Physiological Society and the National Institutes of Health. Rabbits were kept in individual cages in a temperature-controlled room (23°C) and were fed a standard rabbit chow (Harlan Techlab) consisting of 0.29% Na+ and 1.4% K+. For surgical instrumentation (carried out at Univ. of Nebraska), rabbits were anesthetized with a cocktail consisting of 1.2 mg/kg acepromazine, 5.9 mg/kg xylazine, and 58.8 mg/kg ketamine given as an intramuscular injection. Supplemental anesthesia was provided by intravenous pentobarbital sodium at a dose of 1.7 mg/kg as needed. After tracheal intubation, sterile surgery was carried out to implant a pair of piezoelectric crystals across the base of the heart (2 mm; Sonometrics) to evaluate progressive changes in cardiac dimensions over time. At the same time, a stainless steel electrode was secured to the left ventricle and an indifferent electrode was placed in the subcutaneous tissue. The electrodes were exteriorized in the back of the neck for subsequent attachment to a small pacemaker of our own design. The rabbits were allowed to recover from this surgery for 10–14 days before pacing or the sham period was initiated. After the surgery the rabbits were treated for 3 days postoperatively with enrofloxacin (Baytril, 2.3 mg/kg im, twice per day; Miles).
Heart failure model.
The rapid pacing model was used in these studies. In brief, rabbits were paced as previously described (26, 31, 32, 34, 41). After control measurements of cardiac diameter and HR were taken in the awake state, the pacemaker was programmed to 320 beats/min. The animal was paced at this rate for several days to ensure that it would tolerate this level of tachycardia. The rate was gradually increased to between 360 and 380 beats/min over the next 7–10 days and then left at its final rate for 3–4 wk. Cardiac dimensions were recorded once per week with the pacemaker turned off. Animals were instrumented for the final study when their cardiac dimensions had increased by ∼2 mm.
Although plasma ANG II was not measured in the rabbits in this study, we have measured ANG II in normal intact and CHF intact rabbits as part of another study (25). In that study, ANG II was significantly higher in CHF compared with normal rabbits (48.7 ± 7.9 vs 12.8 ± 2 pg/ml; P < 0.05).
Lesions of the area postrema.
The area postrema of rabbits was lesioned (at Univ. of Texas Health Sciences Center, San Antonio) as described earlier (9). In brief, animals were anesthetized with a mixture of ketamine, xylazine, and chlorpromazine (34, 6.5, and 2.5 mg/kg, respectively). The area postrema was exposed by opening the atlantooccipital membrane. Negative pressure was applied to the structure through 25-gauge tubing attached to a suction unit. Animals were postoperatively treated with 1 mg/kg of dexamethasone (LyphoMed) and 75 mg/kg of floxicillin (Aveco). Antibiotic treatment continued for 5 days, and the animals were allowed 14 days to recover.
To confirm an effective lesion, baroreflex testing in response to vasopressin was carried out in each rabbit and compared with the response to an equipressor dose of phenylephrine as described previously (9, 10). In the conscious state the HR response to vasopressin is augmented compared with the response to an equipressor dose of phenylephrine. In intact rabbits, vasopressin evokes an enhancement of baroreflex responses; however, in area postrema-lesioned rabbits the baroreflex bradycardia is similar to that evoked by phenylephrine. This was the case for all lesioned rabbits in this study. For intact rabbits, the phenylephrine baroreflex sensitivity was −2.3 ± 0.27 beats ⋅ min−1 ⋅ mmHg−1vs. −8.7 ± 1.34 beats ⋅ min−1 ⋅ mmHg−1for the vasopressin baroreflex sensitivity (P < 0.005). For lesioned rabbits, the phenylephrine sensitivity was −2.8 ± 0.31 beats ⋅ min−1 ⋅ mmHg−1compared with −3.1 ± 0.46 beats ⋅ min−1 ⋅ mmHg−1for the vasopressin sensitivity (P = not significant). All brains were inspected grossly for evidence of area postrema lesions. Several were sectioned histologically (see Fig.1). Functional and anatomic evidence of effective lesions was observed in all lesioned rabbits.
Hemodynamic and sympathetic nerve recording.
Rabbits were anesthetized with 1.2 mg/kg acepromazine, 5.9 mg/kg xylazine, and 58.8 mg/kg ketamine. After tracheal intubation, sterile surgery was carried out to implant a renal sympathetic nerve electrode and arterial catheter as previously described (26, 32, 34). In brief, a left subcostal incision was made and the kidney was approached in the retroperitoneal space. A bundle of renal nerves were identified and gently freed from surrounding tissue using glass rods. A pair of Teflon-coated stainless steel wire electrodes (0.124-mm OD; A-M Systems) were placed around the dissected renal nerves. To insulate the electrodes and the nerve from the surrounding tissue and to prevent the nerves from desiccation, the electrodes and the nerve assembly were covered with a two-component silicone gel (Wacker Sil-Gel). A ground lead was sutured to the fat close to the electrodes. The electrodes and the ground lead were tunneled beneath the skin to the back and fixed between the shoulder blades. The flank incision was closed.
Through a midline cervical incision, a Micro-Renathane catheter (1.65-mm OD, 0.07-mm ID; Braintree Scientific) was inserted into the left carotid artery for the measurement of arterial pressure and HR. Another catheter was placed in a jugular vein for the measurement of central venous pressure (CVP) and used as a venous access. The catheters were tunneled beneath the skin and brought out at the back of the neck. The catheter was flushed daily with heparin sodium (1,000 U/ml; Elkins-Sinn). After the surgery, the rabbits were treated with antibiotics as described in Lesions of the area postrema.
On the day of the experiment, each rabbit was placed in a Plexiglas box of our own design and attached to the recording equipment. The rabbit was allowed to stabilize for ∼30 min before the experiment began. Several minutes of baseline data were then recorded. Baroreflex curves were generated by measuring the RSNA and HR responses to increases and decreases in arterial pressure by intravenous administration of either phenylephrine or sodium nitroprusside. Administration of phenylephrine (30 μg/kg; American Reagent Laboratories, Shirley, NY) or sodium nitroprusside (100 μg/kg; Hoffman-La Roche, Nutley, NJ) was carried out in random order. MAP was altered at a rate of 1–2 mmHg/s from baseline to peak change. After the control baroreflex curve measurements, the rabbits were given an intravenous injection of the AT1-receptor antagonist L-158,809 (Merck) at a dose of 0.33 mg/kg. This dose completely abolished the response to a 100-ng injection of ANG II. Fifteen to twenty minutes later, baseline measurements were taken again, followed by construction of a second arterial baroreflex curve. Similar experiments were carried out in four groups of rabbits as follows: normal rabbits, normal rabbits with area postrema lesions, CHF rabbits, and CHF rabbits with area postrema lesions.
At the conclusion of the experiment, the rabbits were heparinized and killed with an overdose of pentobarbital sodium. The brain was perfused via a catheter in the ascending aorta as described previously (10). In brief, after the right atrium was punctured, animals were perfused with 1 liter of 0.9% saline, followed by 1 liter of 10% phosphate-buffered Formalin. The brain was removed and placed in a 10% Formalin solution with sucrose for at least 48 h. Frozen sections (40 μm) were cut, mounted, and stained with cresyl violet or by the Kluver-Barrera procedure (22). The sections were then examined for ablation of the area postrema.
HR, RSNA, and MAP data were acquired every 2 s during the changes in arterial pressure evoked by phenylephrine and sodium nitroprusside. A range of pressures were covered to observe the maximum and minimum responses of both HR and RSNA. A sigmoid logistic function was fit to the data using a nonlinear regression program (Sigma Plot v. 4.16, Jandel) run on a Macintosh computer. Four parameters were derived from the following equation Equation 1 whereA is HR or RSNA range,B is the slope coefficient,C is the pressure at the midpoint of the range (BP50), andD is minimum HR or RSNA (21). The peak slope (or maximum gain) was determined by taking the first derivative of the baroreflex curve described by Eq.1 . The first derivative is described byEq. 2 Equation 2All values are expressed as means ± SE. RSNA is expressed as the percentage of maximum nerve activity.
Because of the inherent difficulties in quantifying resting RSNA, we decided to normalize the data to the maximum RSNA achievable for each rabbit. Maximum activity was determined as the response to a bolus injection of sodium nitroprusside that lowered arterial pressure to ∼40 mmHg before administration of the AT1 antagonist. Resting RSNA was expressed as a percentage of the maximum activity. Data were analyzed using a one-way ANOVA for repeated measures when comparing more than two sets of mean data. When the Fratio exceeded the critical value, pairwise comparisons were made using the Student-Newman-Keuls method. A Pvalue of <0.05 was considered statistically significant.
Effectiveness of area postrema lesions.
Figure 1 shows a histological section through the brain stem at the level of the obex. Figure1 A shows an intact rabbit, and Fig.1 B shows an area postrema-lesioned rabbit. The area postrema is clearly ablated in this figure, whereas other structures such as the NTS remain intact. As indicated inLesions of the area postrema, the modulation of baroreflex function by ANG II and vasopressin was abolished in all rabbits with lesions of the area postrema.
Baseline hemodynamics and RSNA.
Figure 2 shows an original tracing of cardiac external diameter before and after several weeks of pacing in an intact rabbit. The average length of pacing in the sham and lesioned rabbits was 20.2 ± 1.8 and 19.2 ± 1.8 days, respectively. As can be seen in Table 1, end-systolic and end-diastolic diameters were significantly increased after pacing (for CHF group only). In addition, the first derivative of cardiac diameter (dD/dt) and fractional shortening were significantly reduced after chronic pacing. There was a reduction in dD/dtof ∼43 and 35% for the CHF and CHF-lesioned groups, respectively. Fractional shortening was reduced ∼42 and 49% in CHF and CHF-lesioned rabbits, respectively. CVP, heart weight, and body weight data are presented in Table 2. CVP was significantly elevated in CHF rabbits both with and without lesions of the area postrema. There were no differences in body weight among the four groups of rabbits. Both heart weight and left ventricular weight were increased in CHF rabbits with and without lesions, as were the ratios of heart weight and left ventricular weight to body weight.
MAP, HR, and RSNA at rest are shown in Table3 for both normal and CHF rabbits. Compared with the normal intact rabbits, CHF intact rabbits exhibited a significantly lower arterial pressure and higher HR both before and after administration of L-158,809. Normal lesioned rabbits had a significantly lower arterial pressure compared with normal intact rabbits before L-158,809. On the other hand, lesioned CHF rabbits had an arterial pressure similar to that of intact CHF rabbits. Arterial pressure fell slightly but significantly in both the intact normal and CHF rabbits after administration of L-158,809. Baseline RSNA was significantly higher in both the intact and lesioned CHF rabbits compared with the respective normal groups both before and after administration of L-158,809. L-158,809 itself had little effect on RSNA in either group.
Baroreflex function: normal rabbits.
Baroreflex function data for intact and lesioned normal rabbits are shown in Figs. 3 and4 and in Table4. In general, baroreflex function was not altered by area postrema lesions. However, there was a significant reduction of the HR range in the normal rabbits after area postrema lesions (P < 0.05). L-158,809 had no significant effect on baroreflex control of HR or RSNA in intact normal rabbits (Fig. 3). Furthermore, as is shown in Fig. 4 and in Table 4, L-158,809 had no effect on baroreflex function in lesioned normal rabbits.
Baroreflex function: CHF rabbits.
In contrast to normal rabbits, intact CHF rabbits had attenuated baroreflex sensitivity for both HR and RSNA. Figure5 shows the mean baroreflex curves for intact CHF rabbits. As can be seen from Table 4, baroreflex sensitivity for HR was 1.6 ± 0.3 beats ⋅ min−1 ⋅ mmHg−1in intact CHF rabbits compared with 5.4 ± 0.7 beats ⋅ min−1 ⋅ mmHg−1for intact normal rabbits (P < 0.001) before administration of L-158,809. Similarly, baroreflex sensitivity for RSNA was 2.3 ± 0.2% of maximum/mmHg for intact CHF rabbits compared with 6.0 ± 0.5% of maximum/mmHg for intact normal rabbits (P < 0.001). Baroreflex sensitivity after administration of L-158,809 was markedly enhanced in intact CHF rabbits for both HR and RSNA, increasing to 4.1 ± 0.7 beats ⋅ min−1 ⋅ mmHg−1for HR and 4.9 ± 0.4% of maximum/mmHg for RSNA (P < 0.02 andP < 0.001, respectively).
Baroreflex function in CHF rabbits with lesions of the area postrema was similar to that in intact CHF rabbits before administration of L-158,809. Baroreflex sensitivity was significantly lower in this group compared with normal rabbits with area postrema lesions (Table 4, Figs.4 and 6). Furthermore, administration of L-158,809 did not augment baroreflex sensitivity for either HR or RSNA in this group of rabbits compared with intact CHF rabbits.
It is now well established that ANG II is capable of reducing arterial baroreflex sensitivity and contributing to sympathoexcitation (4, 16,18, 39, 40). Various CVOs may play a role in mediating the sympathoexcitatory effects of ANG II. The CVO of most prominence in this regard is the area postrema. The area postrema lacks a blood-brain barrier, and therefore, it can “sample” substances in the cerebral intravascular space. ANG II receptors have been demonstrated in various brain stem structures including the area postrema (23), and the application of ANG II to the area postrema evokes a pressor response and sympathoexcitation that can be blocked by the AT1 antagonist losartan (27). Not only can it be shown that ANG II-induced hypertension is reduced in rats with lesions of the area postrema (15), but recent data suggest that transgenic rats that overexpress renin develop hypertension more slowly and to a smaller magnitude when subjected to lesions of the area postrema (1).
The role of ANG II in the pathogenesis of CHF has been extensively investigated (37, 42, 44). This peptide has been implicated in a wide variety of deleterious effects ranging from cardiac remodeling to vasoconstriction to sympathoexcitation. Indeed, the therapeutic management of patients with CHF revolves, to a large extent, around the use of angiotensin-converting enzyme inhibitors and ANG II-receptor antagonists (12, 17, 42, 44). Because the renin-ANG II system is activated in both human and experimental CHF, we reasoned that either elevated circulating ANG II or activation of the brain renin-ANG II system would be capable of contributing to the enhanced sympathetic tone and reduced baroreflex function in the CHF state via an action at the area postrema. The data presented here are the first, to our knowledge, in which the role of the area postrema on baroreflex function in the CHF state has been evaluated. These data strongly suggest that 1) depression of baroreflex sensitivity in CHF is mediated by ANG II and that2) in intact CHF animals the sensitivity of the arterial baroreflex is ANG II dependent. However, in the absence of the area postrema the decreased baroreflex sensitivity in the CHF rabbits is ANG II independent. These data confirm our previous studies (11) and that of DiBona et al. (33, 34), which indicated that AT1-receptor blockade increased arterial baroreflex sensitivity in animals with CHF.
Several important aspects of this study are worthy of note. First, area postrema lesions in normal animals resulted in a significant, albeit small, reduction in MAP (Table 4). This was observed despite the fact that no differences were seen in cardiac dimensions, HR, or RSNA as a result of the lesions. In addition, small but significant differences in MAP were seen after AT1-receptor blockade in the intact normal and CHF groups. Furthermore, no differences in MAP were observed in lesioned CHF rabbits either before or after AT1-receptor blockade. It is not clear why the normal lesioned rabbits exhibited this modest hypotensive effect after area postrema lesions. Others have reported a reduction in MAP in rats with area postrema lesions (7); however, this effect was attributed to a failure of lesioned rats to gain weight and eat normally during a 3-wk recovery period (7, 8). The rabbits used in the present study were used ∼8 wk after area postrema lesioning. There were no differences in body weight between the four groups of rabbits (Table 2). Although we did not monitor food and water intake, there were no apparent differences between normal rabbits and normal lesioned rabbits.
The most marked finding of the present experiment was a complete inhibition of the ability of L-158,809 to enhance arterial baroreflex sensitivity in CHF lesioned rabbits, consistent with previous studies from this laboratory (33, 34). These two studies, along with the present data and that of DiBona et al. (11), provide strong evidence for an important role of ANG II on setting the gain of the arterial baroreflex in the CHF state. The effect of ANG II on baroreflex function is mediated by its ability to cause baroreflex resetting (4,30, 40) and by a reduction in gain (39). By evaluating the response to bolus injections of ANG II in conscious rabbits with lesions of the area postrema, Matsukawa and Reid (30) showed that the attenuation of baroreflex function was mediated via the area postrema. Furthermore, microinjection of ANG II into the area postrema results in an increase in arterial pressure that could be blocked by losartan but not by the AT2 antagonist PD-123319 (27). Although baroreflex sensitivity was enhanced by AT1 blockade in CHF intact rabbits, there was little evidence for resetting in the present experiment as shown by the lack of change in BP50 for either HR or RSNA after administration of L-158,809 (Table 4). It is of note that area postrema lesions did not enhance baroreflex function in CHF rabbits. Rather, they prevented the restoration of baroreflex function by ANG II blockade in CHF rabbits. It can be argued that if ANG II blockade normalizes baroreflex function in CHF and this effect is mediated by the area postrema, then area postrema lesions alone should normalize or at least enhance baroreflex function in the CHF state. It is not clear as to why we did not observe an enhancement in baroreflex function in CHF rabbits with lesions. Clearly, others have shown that area postrema lesions in normal rabbits do not alter baroreflex sensitivity (10, 30) but prevent the modulation of the baroreflex by ANG II. In the setting of CHF, additional mechanisms may operate to depress baroreflex function after area postrema lesions. Because of the relatively chronic nature of the area postrema lesions in this study, it is possible that accessory pathways have developed that maintain a depressed baroreflex function in CHF. This would suggest that basal baroreflex function is somewhat ANG II independent in the CHF state.
Electrophysiological evidence has reinforced the notion that ANG II mediates baroreflex effects via the area postrema. Specific cells within the area postrema respond to application of ANG II with an increase in discharge while others are inhibited (6, 35). Electrical stimulation of the area postrema results in a fall in sympathetic nerve activity that is attenuated by baroreceptor denervation (19, 20). Because neurons from the area postrema project to both the NTS and the rostral ventrolateral medulla (2, 45), it is not surprising that this structure has important modulatory effects on baroreflex function.
The increase in sympathetic outflow in the CHF state is complicated and most likely involves abnormalities at several sites in the peripheral and central nervous systems. In recent studies by Brändle et al. (3) and Levett et al. (24), it was shown that plasma norepinephrine increased to pathophysiological levels in dogs paced into CHF that were subjected to chronic sinoaortic denervation or cardiac denervation, respectively. Therefore, it appears that mechanisms other than abnormalities in these reflexes are responsible for the sustained sympathoexcitatory state in CHF. Another candidate reflex that may be responsible for augmentation of sympathetic tone is the cardiac “sympathetic afferent” reflex (29). Interestingly, in a recent study by Ma et al. (28), it was shown that this sympathoexcitatory reflex was augmented in dogs with pacing-induced CHF. Normalization of the sympathetic afferent reflex sensitivity could be evoked by intracerebroventricular administration of losartan at a dose that had no effect intravenously. From these data and those concerning baroreflex enhancement after AT1blockade, it would appear that some common pathway is responsible for the increase of sympathetic tone by ANG II. We suggest that this common pathway is, in part, the area postrema.
The authors thank Johnnie F. Hackley and Pamela Curry for expert technical assistance.
Address for reprint requests and other correspondence: I. H. Zucker, Dept. of Physiology and Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail:).
This study was supported by National Heart, Lung, and Blood Institute Grant HL-38690. H. Murakami was supported by a Fellowship from the Nebraska Heart Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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