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Am J Physiol Regul Integr Comp Physiol 292: R1137-R1145, 2007. First published October 19, 2006; doi:10.1152/ajpregu.00590.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats

Wei-Zhong Wang, Lie Gao, Yan-Xia Pan, Irving H. Zucker, and Wei Wang

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 19 August 2006 ; accepted in final form 13 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiac "sympathetic afferent" reflex (CSAR) has been reported to increase sympathetic outflow and depress baroreflex function via a central angiotensin II (ANG II) mechanism. In the present study, we examined the role of ANG II type 1 (AT1) receptors in the nucleus tractus solitarii (NTS) in mediating the interaction between the CSAR and the baroreflex in anesthetized rats. We examined the effects of bilateral microinjection of AT1 receptor antagonist losartan (100 pmol) into the NTS on baroreflex control of renal sympathetic nerve activity (RSNA) before and after CSAR activation by epicardial application of capsaicin (0.4 µg). Using single-unit extracellular recording, we further examined the effects of CSAR activation on the barosensitivity of barosensitive NTS neurons and the effects of intravenous losartan (2 mg/kg) on CSAR-induced changes in activity of NTS barosensitive neurons. Bilateral NTS microinjection of losartan significantly attenuated the increases in arterial pressure, heart rate, and RSNA evoked by capsaicin but also markedly (P < 0.01) reversed the CSAR-induced blunted baroreflex control of RSNA (Gainmax from 1.65 ± 0.10 to 2.22 ± 0.11%/mmHg). In 17 of 24 (70.8%) NTS barosensitive neurons, CSAR activation significantly (P < 0.01) inhibited the baseline neuronal activity and attenuated the neuronal barosensitivity. In 11 NTS barosensitive neurons, intravenous losartan effectively (P < 0.01) normalized the decreased neuronal barosensitivity induced by CSAR activation. In conclusion, blockade of NTS AT1 receptors improved the blunted baroreflex during CSAR activation, suggesting that the NTS plays an important role in processing the interaction between the baroreflex and the CSAR via an AT1 receptor-dependent mechanism.

cardiovascular reflexes; losartan; renal sympathetic nerve activity; microinjection; extracellular recording; barosensitivity

THE BAROREFLEX PLAYS A CRUCIAL ROLE in cardiovascular regulation in both normal and disease states (12, 29). Impairment of baroreflex function has been shown to be associated with progression of several diseases and a poor prognosis (10, 14, 23). Recently, depression of baroreflex function in heart failure has been demonstrated to be closely related to enhanced cardiac "sympathetic afferent" reflex (CSAR) function (33). Cardiac sympathetic afferents are known to activate the cardiovascular system, leading to increases in arterial pressure (AP), heart rate (HR), and myocardial contractile function and are activated by metabolic mediators, myocardial ischemia, and cardiac enlargement (18–20). The studies from this laboratory have shown that electrical and chemical stimulation of the CSAR markedly attenuates baroreflex function in normal and heart failure animals (8, 11, 34, 35). Furthermore, the central angiotensin II (ANG II) mechanism has been reported to mediate the interaction between the CSAR and the baroreflex, because intracerebroventricular injection of the ANG II type 1 (AT1) receptor antagonist losartan normalizes the depression of baroreflex function evoked by CSAR activation (8, 9, 38, 39). However, the exact region(s) in the central nervous system responsible for this interaction remain unclear.

The nucleus tractus solitarii (NTS) receives sensory inputs from a vast array of peripheral receptors located on visceral, somatic, and cardiorespiratory organs and would be an effective region for modulating cardiovascular reflexes (1, 29). The NTS is known to be the central termination site of baroreceptors and plays a critical role in integrating the baroreflex (29, 30). On the other hand, axonal tracing studies have shown cardiac sympathetic afferents project to the dorsal horn of the upper thoracic spinal cord through the stellate ganglia and the sympathetic chain (6, 13). The ascending fibers from the dorsal horn of the spinal cord have been demonstrated to terminate in the NTS (22). Electrophysiological evidence also suggests a subgroup of the NTS neurons can be excited by stimulation of cardiac sympathetic afferents (31). Therefore, the NTS has been implicated to be an important area for integrating the central transmission of the CSAR (15, 31).

Although at least two ANG II receptor subtypes, AT1 and AT2, are expressed in rat brain, competition binding studies suggest that only AT1 receptors are found in the NTS (28, 32). It has been suggested that an NTS-ANG II mechanism plays an important role in controlling cardiovascular activity (2, 37). Microinjection of ANG II into the NTS has been found to modify AP and depress baroreflex function, whereas injection of ANG II receptor antagonists facilitates the baroreflex (4, 21). However, no evidence is available to address the question of whether an ANG II mechanism contributes to the interaction between the baroreflex and the CSAR at the level of the NTS.

Hence, the present study was designed to test the role of an NTS-AT1 receptor mechanism in processing of the interaction between the CSAR and the baroreflex in anesthetized rats. We investigated the effects of bilateral microinjection of losartan into the NTS on the blunted baroreflex evoked by CSAR activation. We further determined the effects of CSAR activation on the activity of NTS barosensitive neurons before and after treatment with intravenous injection of losartan.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. All experiments were performed on adult male Sprague-Dawley rats weighing between 310 and 360 g and were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and carried out under the guidelines of the American Physiological Society and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The methods for general surgery, renal sympathetic nerve activity (RSNA) recording, CSAR activation, and baroreflex measurement were described in previous studies by our laboratory (8, 9).

General surgery. Rats were anesthetized with urethane (800 mg/kg ip) and {alpha}-chloralose (40 mg/kg ip). The trachea was cannulated, and the rat was paralyzed with pancuronium bromide (1 mg/kg iv, 0.1 mg/kg thereafter as needed) and ventilated artificially with room air supplemented 100% oxygen. The left common carotid artery was cannulated, and the AP was measured with a pressure transducer (model PT300; Grass Instruments, Quincy, MA) for measurement of mean arterial pressure (MAP). HR was derived from the AP pulse with a PowerLab model 16S (ADInstruments, Colorado Springs, CO). The femoral vein was cannulated for intravenous injections. Rats were placed in a stereotaxic frame (Stoelting, Chicago, IL), and the dorsal surface of the medulla was surgically exposed by incising the atlantooccipital membrane and removing part of the occipital bone and dura. Supplemental doses of {alpha}-chloralose (20 mg/kg iv) were administered to maintain an appropriate level of anesthesia. Body temperature was maintained at ~37°C with an animal temperature controller (ATC1000; World Precision Instruments).

Recording of RSNA. The left renal sympathetic nerves were exposed, identified, dissected free of the surrounding connective tissue, and placed on a pair of platinum-iridium recording electrodes. Both the nerve and the electrodes were covered with a fast-setting silicone (Wacker Sil-Gel). The signal was amplified (band pass 100–1,000 Hz) with a preamplifier (model P 18D; Grass Instruments). The amplified discharge was monitored on a storage oscilloscope (model 121 N; Tektronix, Beaverton, OR), imported to a computer system with other parameters, and then stored on disk until analyzed. Respective noise levels were subtracted from the nerve recording data before percentage changes from baseline were calculated. Integrated RSNA was normalized as 100% baseline in the control period (15).

Activation of the CSAR and the baroreceptors. Epicardial application of capsaicin has been demonstrated to effectively stimulate the cardiac sympathetic afferents (9, 39). The chest was opened through the fourth intercostal space. The pericardium was removed to expose the left ventricle. A filter paper containing capsaicin (3 x 3 mm, 0.4 µg in 2 µl) was applied to the anterior surface of the left ventricle. The baroreflex was tested about 2 min after capsaicin was applied, and then the epicardium was rinsed three times with 10 ml of warm normal saline (38°C). The time interval of repeated capsaicin was at least 30 min to allow the AP, HR, RSNA, and discharge of NTS neurons to return to, and stabilize at, their control levels. To test and evaluate the barosensitivity of NTS neurons in neuronal recording experiments, we placed a vascular occluder (Harvard Apparatus) around the descending thoracic aorta above the diaphragm, to elevate AP by constricting the aorta.

Microinjections into the NTS. Microinjections were made from four-barrel micropipettes with total tip diameters of 20–30 µm and performed using a four-channel pressure injector (PM2000B; World Precision Instruments). The injections were made over a 10-s period, and a 50-nl injection volume was measured by observing the movement of the fluid meniscus along a reticule in a microscope. The dorsal medial NTS (coordinates in mm with respect to calamus scriptorius: 0.4–0.5 rostral, 0.5–0.6 lateral, and 0.4–0.5 deep) was identified by injecting L-glutamate (2 nmol) and observing a depressor response of at least 25 mmHg. Losartan and L-glutamate were dissolved in artificial cerebrospinal fluid (aCSF). The time interval between bilateral injections was within a 60-s period. At the end of the experiments, 50 nl of 2% Pontamine sky blue were injected for marking the injection sites.

Extracellular recording of the NTS neurons. A single-unit extracellular recording was obtained using a single micropipette (tip diameter 1–2 µm; resistance 5–12 M{Omega}) filled with 0.5 M sodium acetate dissolved in 2% Pontamine sky blue. The electrode was advanced using a microdrive (HSE-HA 864/1) into the dorsal medial NTS at a speed of 1–2 µm/s until single-unit activity was recorded. The spontaneous action potentials were amplified using a high-impedance preamplifier (Dagan; band pass 100–3,000 Hz) and fed into a window discriminator (Mentor N-750 spike analyzer), which generates a standard pulse for each spike. Potentials were visualized on an oscilloscope (model 121N; Tektronix). The pulse output of the discriminator was then fed into a rate/interval monitor (HFC) whose analog output is proportional to the number of spikes per unit time (1 s). These signals were displayed online on the computer and recorded on the data acquisition system.

The barosensitivity of NTS neurons was identified by their excitatory response to transient AP elevation (20–50 mmHg) by aorta occlusion (5–10 s). Baroreceptor stimulation by aorta occlusion or balloon inflation has been performed to conveniently identify the barosensitivity of neurons in the brain stem (7, 16). Some specific NTS barosensitive neurons that were inhibited by aorta occlusion were not further observed in the present study. After a neuron of interest was recorded, a small amount of 2% Pontamine sky blue was iontophoresed (–15 µA, 10 min) to mark the recording location for histological analysis.

Histological analysis. At the end of the experiment, the rat was given a lethal injection of pentobarbital sodium (100 mg/kg iv) and perfused with 10% formaldehyde solution (100 ml) intracardially. The brain stem was then quickly removed and fixed in 10% buffered Formalin. Frozen 50-µm coronal sections were made on a freezing microtome and mounted on slides. The dye spot and spread area for injection or cell recording sites were identified and plotted on standardized sections according to the atlas of Paxinos and Watson (24). Data were excluded if the injection or recording sites were not located in the medial NTS area. Figure 1 shows the locations of the microinjection sites (A) and the recorded neurons (B) in the NTS. There was no clear difference in topographical distributions between barosensitive and nonbarosensitive neurons within the NTS.


Figure 1
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  		Fig. 1. Distributions of the microinjection (A) and recording sites (B) in the nucleus tractus solitarii (NTS). bullet, Microinjection sites; {blacksquare}, barosensitive neurons; {square}, nonbarosensitive neurons. AP, area postrema; Gr, gracile nucleus; Cu, cuneate nucleus; Sol, NTS; 10, motor nucleus of vagus nerve; 12, nucleus of hypoglossal nerve.

 
Construction of baroreflex curves and evaluation of barosensitivity of NTS neurons. Baroreflex curves were generated by measuring the RSNA responses to a decrease and an increase in AP by intravenous injection of either nitroglycerin (25 µg) or phenylephrine (10 µg). MAP was altered at a rate of 1–2 mmHg/s during these infusions. Increases and decreases in MAP were carried out in random order. The MAP data were acquired every 2 s from the threshold to the saturation points. A sigmoid logistic function was fit to the data using a nonlinear regression program (SigmaPlot version 8.0; SPSS) run on a computer. Four parameters were derived from the following equation: %RSNA = A/{1 + exp[B(MAP – C)]} + D, where A is the RSNA range, B is the slope coefficient, C is the pressure at the midpoint of the range (BP50), and D is minimum RSNA. The peak slope [or maximum gain (Gainmax)] was determined by taking the first derivative of the baroreflex curve described by the equation.

The barosensitivity of NTS neurons was evaluated as percent changes of baseline discharge during aorta occlusion. The magnitude of barosensitivity of NTS neurons in the control period was normalized as a barosensitivity of 100%. The barosensitivity was evaluated again after treatments at the same AP elevation used during the control test. Decreases or increases in barosensitivity were expressed as a percent reduction or elevation from the magnitude of barosensitivity in the control period. Importantly, we also examined the change in relationship between neuronal discharge and AP during aorta occlusion (spikes/mmHg) for further evaluation of the barosensitivity.

Statistical analysis. The data are presented as means ± SE. We constructed composite baroreflex curves by averaging the four parameters of the logistic equation for all curves and using the mean parameters to construct a single curve. The changes in integrated RSNA and neuronal activity after treatments were evaluated as percent changes from control because of the variability in baseline RSNA in each animal and discharge in each neuron. Comparisons between control and experimental interventions were made using either Student's t-test (paired and unpaired) or one-way analysis of variance. Differences were considered to be statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of bilateral microinjection of losartan into the NTS on cardiovascular parameters in response to epicardial application of capsaicin. Table 1 summarizes the cardiovascular parameters, including AP, HR, RSNA, and the baroreflex before and 10 min after NTS injection of losartan (100 pmol each side) or aCSF (50 nl each side). NTS losartan markedly enhanced Gainmax for baroreflex control of RSNA (from 1.98 ± 0.10 to 2.55 ± 0.15%/mmHg, P < 0.01, n = 8) without altering baseline AP, HR, and RSNA. In control tests, NTS injection of 50 nl of aCSF (n = 5) did not alter any cardiovascular parameters. Figure 2 presents the baroreflex control of RSNA (MAP-RSNA curves) before and after NTS injection of losartan. As shown, baroreflex sensitivity was increased following NTS losartan administration.


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  		Table 1. Effects of losartan microinjected into the NTS on MAP, HR, RSNA, and baroreflex parameters before and after epicardial application of capsaicin

 

Figure 2
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  		Fig. 2. Effect of NTS microinjection of losartan on baroreflex control of renal sympathetic nerve activity (RSNA). Average logistic function regression curves were generated before and after bilateral microinjection of losartan into the NTS (n = 8). MAP, mean arterial pressure. Inset: average gain of these mean baroreflex curves. *P < 0.01 vs. control.

 
Figure 3 shows original representative tracings of AP and RSNA during epicardial application of capsaicin (0.4 µg) before and 10 min after NTS microinjection of losartan. Epicardial application of capsaicin produced significant (P < 0.01) increases in AP, HR, and RSNA but markedly attenuated (P < 0.01) the baroreflex control of RSNA (Table 1 and Figs. 4 and 5). The peak changes in AP and HR were reached within 60 s and gradually recovered to the control level. The maximal change in RSNA was reached within 30 s and returned to the control level within 60 s. The baroreflex test was performed 2 min after capsaicin treatment, when the variables of AP and RSNA had returned to their control values. Capsaicin had no significant effects on the range of RSNA response, BP50, and minimum RSNA, but it did reduce the average slope and Gainmax of the baroreflex curve. The increases in AP, HR, and RSNA were significant, and the decrease in baroreflex sensitivity evoked by capsaicin was significantly (P < 0.01) prevented 10 min after NTS losartan injection compared with NTS injection of aCSF (n = 5) (Figs. 4 and 5). It was further demonstrated that, 10 min after NTS losartan, the baroreflex sensitivity during capsaicin completely recovered to the control level (Gainmax: 2.22 ± 0.11 vs. 2.55 ± 0.15%/mmHg, P > 0.05) (Table 1 and Fig. 5B).


Figure 3
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  		Fig. 3. Original tracings showing the responses of arterial pressure (AP) and RSNA to epicardial application of capsaicin (0.4 µg) before and after bilateral microinjection of losartan (100 pmol) into the NTS. These data were taken from 1 animal. The NTS was functionally identified by the rapid depressor response to injection of L-glutamate (2 nmol).

 

Figure 4
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  		Fig. 4. Bar graphs showing the magnitude of changes ({Delta}; means ± SE) in MAP (A), RSNA (B), average slope (C), and maximum gain (Gainmax; D) during epicardial application of capsaicin before and after microinjection of losartan or artificial cerebrospinal fluid (aCSF) into the NTS. *P < 0.01 vs. aCSF.

 

Figure 5
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  		Fig. 5. Composite baroreflex curves showing the effect of epicardial application of capsaicin on baroreflex control of RSNA before (A) and after (B) microinjection of losartan into the NTS. Inset: average gain of these mean baroreflex curves. *P < 0.01 vs. control.

 
Effects of epicardial application of capsaicin on activity of NTS neurons. A total of 53 spontaneously discharging neurons within the NTS were recorded from 28 rats in this study. Of 53 units, 35 were functionally identified as barosensitive neurons because they were rapidly excited by 110.5 ± 13.1% (P < 0.01) when AP was transiently elevated (34.4 ± 1.7 mmHg) by aorta occlusion. The remaining 18 units without responsiveness to the same AP elevation (37.8 ± 2.1 mmHg) were assumed to be nonbarosensitive neurons. There was no significant difference in baseline discharge between baro- and nonbarosensitive groups (5.4 ± 0.6 vs. 4.8 ± 0.7 spikes/s, P > 0.05).

Figure 6 shows original representative tracings of baroreflex activation by aorta occlusion and the responses of a barosensitive NTS neuron to epicardial application of capsaicin. The magnitude of AP elevation during aorta occlusion before and after treatments was similar. This magnitude of AP elevation (35.7 ± 3.2 mmHg, n = 8) in several rats was verified to effectively activate the baroreflex control of HR and RSNA (Fig. 6A). In 17 of 24 (70.8%) barosensitive NTS neurons (4.9 ± 0.7 spikes/s), the resting discharge was significantly (P < 0.05) inhibited by 35.6 ± 4.0% during epicardial capsaicin application. In these neurons, the barosensitivity was also markedly (P < 0.01) attenuated to 33.5 ± 6.8% ({Delta}discharge/{Delta}AP: from 0.142 ± 0.022 to 0.032 ± 0.006 spikes/mmHg) (Fig. 7). The remaining seven (29.2%) barosensitive NTS neurons (5.4 ± 0.9 spikes/s) did not respond to capsaicin. In contrast, neither discharge (4.8 ± 1.3 vs. 5.0 ± 1.5 spikes/s, P > 0.05) nor barosensitivity ({Delta}discharge/{Delta}AP: 0.137 ± 0.038 vs. 0.126 ± 0.028 spikes/mmHg, P > 0.05) in seven capsaicin-sensitive neurons was changed during epicardial application of 0.9% normal saline (2 µl).


Figure 6
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  		Fig. 6. Original tracings showing the response of baroreflex function to aorta occlusion (A) and the responses of a barosensitive neuron in the NTS (B) to epicardial application of capsaicin.

 

Figure 7
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  		Fig. 7. Bar graphs showing the responses of discharge (A) and barosensitivity (B) of NTS barosensitive neurons to epicardial application of capsaicin (n = 17, solid bars) and normal saline (NS; n = 7, open bars). Values are means ± SE. *P < 0.01 vs. baseline.

 
In addition, we also tested the response of nonbarosensitive NTS neurons to epicardial capsaicin. Interestingly, in response to capsaicin, it was found that of 18 nonbarosensitive neurons, 10 (56%) were excited by an average of 71.8%, 3 (16%) were inhibited by an average of 48.3%, and 5 (28%) did not respond.

Effects of intravenous losartan on the NTS neuronal barosensitivity in response to epicardial application of capsaicin. Eleven barosensitive NTS neurons from 11 rats that were sensitive to capsaicin were tested in this experiment. Figure 8 shows the responses of neuronal barosensitivity to epicardial application of capsaicin 10 min after intravenous losartan (2 mg/kg in 0.3 ml normal saline). It was found that intravenous injection of losartan significantly decreased AP (from 96 ± 2 to 82 ± 3 mmHg, P < 0.01) and HR (from 368 ± 7 to 332 ± 7 beats/min, P < 0.01), whereas it increased ongoing activity of barosensitive neurons by 30.1% (from 6.0 ± 1.2 to 7.5 ± 1.4 spikes/s, P < 0.05). The neuronal barosensitivity was increased to 119 ± 9% ({Delta}discharge/{Delta}AP: from 0.124 ± 0.018 to 0.159 ± 0.011 spikes/mmHg, P < 0.05) 7 min after intravenous losartan. Ten minutes after losartan, capsaicin did not significantly decrease the baseline discharge (7.3 ± 1.3 vs. 6.4 ± 1.1 spikes/s, P > 0.05) or barosensitivity ({Delta}discharge/{Delta}AP: 0.159 ± 0.011 vs. 0.144 ± 0.014 spikes/mmHg, P > 0.05). We also noted that the baseline AP and neuronal discharge between 7 and 10 min after losartan were similar. The magnitudes of percent changes in discharge and barosensitivity of NTS neurons during capsaicin application before and after intravenously injected losartan are shown in Fig. 8B. In addition, intravenous normal saline (0.3 ml, n = 3) did not modify capsaicin-induced inhibition of NTS neurons (data not shown).


Figure 8
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  		Fig. 8. Effects of intravenous injection of losartan on activity of NTS barosensitive neurons during epicardial application of capsaicin. A: original tracings showing the effects of intravenous losartan (2 mg/kg) on AP and activity in an NTS barosensitive neuron. B: bar graph showing the percent changes in discharge and barosensitivity of NTS barosensitive neurons (n = 11) during epicardial application of capsaicin before and after intravenous losartan (2 mg/kg). Values are means ± SE. *P < 0.05 vs. control (before losartan).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Two important observations were made from the present study: 1) in the NTS, local blockade of AT1 receptors attenuated the increased sympathetic activity and the blunted baroreflex evoked by CSAR activation; and 2) at the level of the NTS barosensitive neuron, CSAR activation produced a reduction of both baseline discharge and barosensitivity, which was effectively abolished by systemic losartan. The current data suggest that endogenous ANG II acting at AT1 receptors in the NTS plays an important role in mediating the interaction between the baroreflex and the CSAR.

In chronic heart failure, the CSAR has been reported to be augmented, and its enhancement may be one important factor that contributes to sympathetic hyperactivity and baroreflex impairment (8, 9, 33–35). The current data also show that in normal rats, chemical activation of the CSAR by application of capsaicin to the left ventricle produced a sympathoexcitation and a reduction in baroreflex control of RSNA, which was consistent with the previous studies (8, 9, 34). Consistent with our previous study (9), we noted that CSAR activation by capsaicin significantly attenuated the values of slope and maximum gain of baroreflex curve without affecting the values of range of RSNA response and minimum RSNA. Because increases in AP and RSNA after capsaicin usually recovered to control levels within ~2 min, prebaseline levels of baroreflex testing before and after capsaicin were similar. This may explain why the baroreflex curves after capsaicin are not significantly shifted right and upward. The baroreflex test was conducted 2 min after capsaicin treatment, when the variables of AP and RSNA had returned to their control values, which indicates that at this time the effects of capsaicin had dissipated. Although we also have observed a significant attenuation of baroreflex sensitivity ~2 min after capsaicin treatment, the observed effects on the baroreflex may be less than those that would have occurred at an earlier time. To address the question of the central mechanism responsible for interaction between the CSAR and the baroreflex, there are at least two essential factors to understand. These are to identify the integrative region(s) in the brain and the neurotransmitters/receptors that participate in this interaction. Because the NTS receives convergent inputs from baroreceptor afferents and cardiac sympathetic afferents (22, 31) and the NTS ANG II mechanism participates in integrating the baroreflex (4, 5), we chose the NTS as the target region where the interaction between the CSAR and the baroreflex may occur.

The present results from the microinjection experiments showed that AT1 receptor blockade in the NTS significantly increased the baroreflex function without modifying baseline AP, HR, and RSNA. This observation is consistent with previous studies from other laboratories (5, 21). These data suggest that endogenous ANG II acting on the NTS AT1 receptors modifies baroreflex function. Importantly, we also found that AT1 receptor blockade in the NTS significantly attenuated the increases in AP, HR, and RSNA evoked by capsaicin and also effectively reversed the attenuation of the baroreflex by capsaicin. We also observed that the capsaicin-induced barosensitivity after NTS losartan not only was reversed to the preinjection level but also was enhanced (Fig. 5B). Therefore, we believe that NTS losartan abolishes capsaicin-induced depression of baroreflex function, as well as increasing baroreflex sensitivity. These data strongly indicate that the functional states of the NTS AT1 receptors are very important in mediating both the central transmission of the CSAR and the interaction between the CSAR and the baroreflex. The present data suggest that during CSAR activation, endogenous ANG II release and action on NTS AT1 receptors results in an increase in sympathetic outflow and attenuation of the baroreflex. However, no direct evidence is available to demonstrate that, at the NTS level, release of ANG II is increased and the function of AT1 receptors is upregulated when the CSAR is activated. Importantly, previous data support this idea. For instance, 1) angiotensin-converting enzyme mRNA expression within the NTS is higher in heart failure rats (27); and 2) AT1 receptor density in the NTS is upregulated in a rabbit with heart failure in a radioautographic study (40). Although the CSAR is significantly augmented in heart failure (33, 34), contribution of the CSAR enhancement to increased ANG II or upregulated AT1 receptors in the NTS has not been confirmed and awaits further study. In addition to the NTS, other cardiovascular centers such as the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus also may participate in this interaction (16, 38).

To further elucidate the role of NTS-AT1 mechanism in this interaction, we directly observed the effect of CSAR activation on NTS barosensitive neurons. In this work, we did not electrically stimulate the aortic or carotid sinus nerve to identify NTS neurons receiving baroreceptor afferents, and clearly, this may be considered a limitation. We believe that AP elevation by aorta occlusion may be similar to AP elevation by intravenous injection of phenylephrine, which has been shown to activate NTS neurons that are driven by single-pulse stimulation of the aortic nerve (26). In the present study, an average of ~35 mmHg of AP elevation during aorta occlusion was verified to effectively stimulate the baroreceptors and activate the baroreflex (Fig. 6A). Moreover, we also found that in some tested NTS neurons, there existed a significantly positive correlation (r = 0.76; data not shown) between the change in AP by aorta occlusion and the firing rate of NTS neurons. In addition, a large change in AP (>50 mmHg) during recording of single units often resulted in the loss of the recording, because the distance between the stationary microelectrode and the neuron of interest increased.

At the level of NTS barosensitive neurons, CSAR activation inhibited the basal discharge rate and attenuated the barosensitivity, which could be prevented by intravenous losartan. Losartan has been demonstrated to cross the blood-brain barrier in rats (17). Because losartan was given intravenously in this work, its exact targets could not be identified. However, some important observations from the current data indicate that endogenous ANG II acted at AT1 receptors to mediate the CSAR-induced decreases in baseline discharge and barosensitivity of NTS neurons. We believe that the inhibitory effect of capsaicin on NTS neurons is not due to neuronal baroreflex activation, because AP elevation by CSAR activation will produce an excitation of NTS barosensitive neurons. It is well known that NTS barosensitive neurons, which receive input from the peripheral baroreceptors, excite the caudal ventrolateral medulla (CVLM) neurons via a glutamatergic projection, whereas the CVLM neurons inhibit the RVLM vasomotor neurons via a GABA-ergic projection (1, 29). The RVLM has been recognized as a critical site for generating sympathetic outflow and controlling baroreflex function (29). Therefore, it is logical to assume that the inhibitory effect of CSAR activation on NTS barosensitive neurons would increase sympathetic outflow and that attenuation of the barosensitivity of NTS neurons would decrease the baroreflex function. Therefore, these new findings contribute to our understanding of the mechanism responsible for CSAR-induced sympathetic hyperactivity and baroreflex impairment. Because there are several pathways between the NTS and RVLM, we guess that reduction of barosensitive neurons by CSAR activation may not be a unique pathway (NTS-CVLM-RVLM pathway) to cause sympathoexcitation. More recently, we furthermore found that CSAR activation significantly excites the chemosensitive neurons within the commissural NTS, where neurons directly send the excitatory projection to the RVLM (36). Therefore, in addition to inhibition of barosensitive neurons, excitation of chemosensitive neurons may be another important mechanism within the NTS to mediate the CSAR-induced sympathoexcitation. Taking these findings together, we think that several mechanisms may simultaneously and together mediate the processing of CSAR-induced sympathoexcitation.

It has been reported that a neuron group exists within the NTS that can be excited by stimulation of cardiac sympathetic afferents and that excitatory amino acids may mediate this central transmission (15, 31). Because these NTS neurons have not been further identified by barosensitivity, we assume these neurons directly receive cardiac sympathetic afferent input rather than baroreceptor input. Our results provide evidence for this possibility, because a majority (56%) of nonbarosensitive neurons were excited by epicardial capsaicin. Therefore, it is possible that the CSAR-excitatory neurons are interneurons that release inhibitory neurotransmitters. CSAR-induced excitation of this inhibitory interneuron may produce an inhibition of NTS barosensitive neurons, which is consistent with CSAR activation resetting barosensitivity. There is substantial evidence that GABA may be the inhibitory neurotransmitter within the NTS. For example, iontophoretic application of GABA markedly reduced or abolished the responses of NTS neurons to stimulation of baroreceptors (3). Inhibitory interneurons in the NTS have become a well-accepted notion to explain baroreflex resetting (7, 25).

In summary, blockade of AT1 receptors significantly improved the depressed baroreflex function evoked by CSAR activation at the level of the NTS in anesthetized rats, suggesting that an NTS-ANG II mechanism plays an important role in processing the interaction between the baroreflex and the CSAR. However, because of the acute nature of the experiments in the present study, it is not known whether the NTS-ANG II mechanism exists in more chronic pathological conditions such as chronic heart failure.


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This study was supported by National Heart, Lung, and Blood Institute Grant R01 HL-077691 and PO1 HL-62222.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Wang, Dept. of Cellular and Integrative Physiology, Univ. of Nebraska Medical Center, Omaha, NE 68198-5850 (e-mail: weiwang{at}unmc.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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