AJP - Regu Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 280: R1665-R1673, 2001;
0363-6119/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shigematsu, H.
Right arrow Articles by Takeshita, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shigematsu, H.
Right arrow Articles by Takeshita, A.
Vol. 280, Issue 6, R1665-R1673, June 2001

Endogenous angiotensin II in the NTS contributes to sympathetic activation in rats with aortocaval shunt

Hideaki Shigematsu, Yoshitaka Hirooka, Kenichi Eshima, Miwako Shihara, Tatsuya Tagawa, and Akira Takeshita

Department of Cardiovascular Medicine, Cardiovascular Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812 - 8582, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recent studies have suggested that the central nervous system is responsible for activation of sympathetic nerve activity (SNA) and the renin-angiotensin system in heart failure (HF). The aim of this study was to determine whether activation of the renin-angiotensin system within the nucleus of the solitary tract (NTS) plays a role in enhanced SNA in HF. High-output HF was induced by an aortocaval (A-V) shunt with some modifications in the rat. These rats exhibited a left ventricular dilatation and hemodynamic signs of high-output HF. Urinary catecholamine excretion and maximal renal SNA (RSNA) were greater in the A-V shunted rats than in the control rats. Microinjection of an angiotensin II type 1-receptor antagonist, CV11974, into the NTS was performed. The arterial pressure and RSNA were reduced by CV11974 to a greater degree in the A-V shunted rats than in the control rats. The expression of angiotensin-converting enzyme mRNA in the medulla was greater in the A-V shunted rats than in the control rats. These results suggest that activation of the renin-angiotensin system within the NTS contributes to an enhanced SNA in this model.

arteriovenous fistula; brain; sympathetic nervous system; heart failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC HEART FAILURE (HF) is characterized by an enhanced neurohumoral drive in experimental animals as well as in patients (32, 34, 52). In patients with chronic HF, plasma norepinephrine levels increase with the severity of HF and the mortality rate (41). Although several mechanisms have been proposed, the actual mechanisms responsible for this sympathoexcitation in HF are not known. Impaired arterial baroreceptor reflex function has been suggested as one of the mechanisms responsible for an increased sympathetic tone in HF (32, 52). However, recent studies have shown that this increase in basal sympathetic nerve activity (SNA) in HF is difficult to explain based only on this mechanism (5, 27). Therefore, central nervous system mechanisms have been suggested to be involved in this sympathoexcitation in HF (52).

The treatment of chronic HF with angiotensin-converting enzyme (ACE) inhibitors or beta -blockers has been shown to decrease the mortality of the patients (32, 34). In patients with HF, the renin-angiotensin system is also activated (32, 34). Activation of the sympathetic nervous system and the renin-angiotensin system in HF contributes to excessive vasoconstriction (32, 34, 52). Although activation of the renin-angiotensin system plays an important role in HF (31), the depressed baroreflex contol of the heart rate or renal SNA (RSNA) may be restored by blocking the ANG II type 1 (AT1) receptor (11, 37, 38). ANG II modulates sympathetic function at many loci in the central and peripheral nervous systems. DiBona et al. (11) have shown that intracerebroventricular as well as intravenous administration of losartan improves the depressed arterial baroreflex control of RSNA in HF produced by myocardial infarction in rats. Their results indicate that ANG II within the brain contributes to an enhanced sympathetic drive in HF. However, the regions in the brain that are activated by ANG II in HF are not known. The nucleus of the solitary tract (NTS) plays an important role in controlling SNA and contains many AT1 receptors (2, 18, 47, 48). For example, microinjection of ANG II into the NTS has been found to elicit a pressor response (8). On the other hand, microinjection of an ANG II-receptor antagonist, [Sar1,Thr8]ANG II, into the NTS has been shown to facilitate reflex control of heart rate (7). Evidence suggests that the NTS contains all of the components of the renin-angiotensin system including angiotensinogen, ACE, ANG II, and AT1 receptors (2, 4, 9, 18, 29, 35, 46-48). However, questions arise as to whether the brain renin-angiotensin system in the NTS is altered in HF, which would, in turn, contribute to an enhanced sympathetic drive in HF.

The aim of this study was to examine the role of endogenous ANG II in the NTS in sympathoexcitation in HF. For this purpose, we micoinjected an AT1-receptor antagonist into the NTS and examined resultant changes in the arterial blood pressure, heart rate, and RSNA in rats with high-output HF resulting from the creation of aortocaval (A-V) shunts with some modification to increase the shunt flow (20). The A-V shunt model has been used for the study of circulatory overload state, which, in turn, produces a high-output HF such as an inadequate blood flow supply to organs (1, 6, 15, 17, 23, 45). Moreover, it has been shown that neurohumoral activation has been shown to occur in this model (15, 45). From these observations, the effects of AT1-receptor blockade within the NTS on the sympathetic nervous system in this model of high-output HF were examined.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Wistar-Kyoto rats weighing 300-350 g were obtained from an established colony at the Animal Research Institute of Kyushu University Faculty of Medicine (39). This study was reviewed by the Committee on Ethics in Animal Experiments of Kyushu University and was performed in accordance with the Guidelines for Animal Experiments of Kyushu University.

Aortocaval shunt model. An arteriovenous (A-V) shunt was induced using procedures described previously (15, 17, 23). Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the inferior vena cava and abdominal aorta were exposed through a midline abdominal incision under sterile conditions. The A-V shunt was produced using an 18-gauge needle at a point ~5 mm caudal to the left renal vein. The patency of the shunt was verified visually on the basis of swelling of the vena cava and mixing of arterial blood with venous blood. To increase the shunt flow, a constriction of the abdominal aorta was performed at a point ~5 mm caudal to the shunt as described previously (20). Sham operations were performed in control rats using procedures identical to those used for the A-V shunted rats, except for insertion of the needle and aortic constriction. The abdominal incision was closed, and the animal was allowed to recover. Six to eight weeks after the surgery, the rats were anesthetized with pentobarbital sodium (30 mg/kg ip), and an echocardiographic examination of left ventricular (LV) diameter and wall motion was performed with a 7.5-MHz probe (ALOKA, SSD-5500). Short-axis views were used to guide the cursor for M-mode images of the LV. LV diastolic dimensions were measured from the trailing edge of the intraventricular septum to the leading edge of the posterior wall at the point of maximal ventricular diameter. Systolic dimensions were taken at the minimum ventricular diameter of the beats in systole using the same edge definition. Systolic ventricular function was quantified as ejection fraction. Urinary catecholamines were measured by high-performance liquid chromatography.

For microinjection experiments, anesthesia was induced with pentobarbital sodium (50 mg/kg ip), and an intravenous infusion of propofol (20~30 mg · kg-1 · h-1) was begun as the effect of the pentobarbital sodium diminished, as described previously (13, 25). The adequacy of anesthesia was verified by the absence of a withdrawal response to nociceptive stimulation of a hindpaw and by a stable baseline arterial pressure and heart rate. The femoral artery and vein were cannulated for recording arterial blood pressure and for the administration of drugs. The anesthetized rats were artificially ventilated.

Recording of the efferent renal SNA. The left kidney was exposed through a left retroperitoneal flank incision. A branch of the renal nerve was isolated from the fat and connective tissues. A pair of stainless steel bipolar electrodes was placed beneath the renal nerve, and the renal sympathetic nerve around the recording electrode was covered with silicone gel (Sil-Gel, Wacker-Chemie). Multifiber renal SNA (RSNA) was recorded and preamplified with a high-gain differential amplifier (band width 150-1,000 Hz; model MEG-1100, Nihon-Kohden) fed into a nerve traffic analyzer (model MET-1100, Nihon-Kohden) and converted to spikes by a window discriminator, as described previously (13, 19). Background noise levels were determined by an intravenous injection of hexamethonium (30 mg/kg) and, thereafter, an intravenous injection of an overdose of pentobarbital sodium (100 mg/kg). The background noise levels were subtracted during a later analysis of RSNA (49). The baseline RSNA was recorded at resting conditions as well as during infusion of sodium nitroprusside (10 µg · kg-1 · min-1), which decreased the arterial pressure by ~40 mmHg and increased RSNA to maximal levels (43). The resting RSNA was normalized to the maximal RSNA as determined above. For analysis of changes in RSNA in the multifiber preparation, RSNA was normalized to 100% as the stable value before microinjection. This method has been used to normalize the differences derived from electrode position, which may influence the results in the multifiber recordings (14).

Microinjection study. Sinoaortic denervation and vagotomy were performed before the microinjection study to eliminate arterial and cardiopulmonary baroreceptor reflexes, as described previously (13, 21). The rat was placed in a stereotaxic frame with the head flexed downward to an angle of approximately 45°. The dorsal surface of the medulla was exposed after a midline incision through the skin. A unilateral microinjection into the NTS was made via a glass micropipette (OD 50-70 µm), which was connected to a Hamilton microsyringe. The tip of the glass micropipette was lowered 0.5 mm below the surface of the medulla to coordinates 0.6 mm rostral and 0.6 mm lateral with respect to the calamus scriptorius, as described previously (8, 13, 42). The volume of vehicle and drug injections was 50 nl and was administered over a 30-s period. Artificial cerebrospinal fluid (CSF) at pH 7.4 was used as vehicle.

L-Glutamate (500 pmol in 50 nl) was microinjected unilaterally into the NTS to identify the depressor site. An AT1-receptor antagonist, CV11974, was microinjected either unilaterally into the NTS or into the area postrema at doses of 50 and 500 pmol in 50 nl. After over 30 min of the microinjection of CV11974, the ANG II type 2 (AT2)-receptor antagonist, PD123319, was microinjected into the NTS. As a control, we microinjected the vehicle solution used to dissolve CV11974 into the NTS to exclude the nonspecific effects.

RNA isolation and Northern blot analysis. Four rats with an A-V shunt and four control rats were killed under deep pentobarbital sodium anesthesia, and the lower brain stem (0.2 mg, 2 mm thick) including the NTS was removed and immediately frozen in liquid nitrogen. Total RNA was prepared from the brain stem using ISOGEN (Nippon Gene) followed by poly(A)+RNA purification with an oligo(dt)-cellulose column (Takara Shuzo). Poly(A)+RNA (5 µg/lane) was separated by electrophoresis and transferred onto a nylon membrane (Hybonde N+, Amersham). Hybridization was carried out using a 32P-labeled EcoR V-EcoR I fragment of rat ACE cDNA followed by autoradiography (12, 13, 24). Autoradiograms were scanned using a Fuji phosphoimaging system to measure the levels of ACE-receptor mRNAs, which were normalized against those of GAPDH mRNA.

Histological examination. To determine the site of injection by postmortem examination after the completion of the experiments, 100 nl of Evans blue were injected through a micropipette positioned at the site of drug injection. After the Evans blue injection, the rat was deeply anesthetized with an overdose of pentobarbital sodium and the brain was perfused through the heart with saline followed by 4% paraformaldehyde solution. The brain stem was removed, and frozen sections (50 µm) were cut serially. As shown in Fig. 1, the location of the injection site was identified by microscopic examination.


View larger version (131K):
[in this window]
[in a new window]
  		Fig. 1.   Photomicrograph of a section through the nucleus of the solitary tract (NTS), which was stained by neutral red, showing the injection site. The arrowhead indicates the injection site. Scale bar represents 500 µm on the photomicrograph. AP, area postrema; X, dorsal nucleus of vagus; XII, hypoglossal nucleus.

Statistical analysis. All values are expressed as means ± SE. The unpaired t-test was used to compare baseline values between the groups. The paired t-test was used to examine the effects of each intervention within a group. A two-way ANOVA followed by Bonferroni's multiple-comparison test was used to compare the differences in changes in arterial pressure, heart rate, and RSNA between the groups and between the drug dosages. Differences were considered significant for values of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline hemodynamic and echocardiographic evaluation. Table 1 shows the results of the hemodynamic and echocardiographic examination of the A-V shunted rats and the control rats. The mean arterial pressure (MAP) and heart rate did not differ between the two groups. However, the LV end-diastolic pressure (LVEDP) was higher (P < 0.0001) and the LV dP/dt lower (P < 0.05) in the A-V shunted rats than in the control rats. In addition, both the LVEDP and the LV end-systolic diameters were larger (P < 0.0001 for each), and the ejection fraction was smaller (P < 0.001) in the A-V shunted rats than in the control rats. Finally, the calculated stroke volume and cardiac output were significantly higher in the A-V shunted rats than those in the control rats.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Hemodynamic and echocardiographic values in the control and the A-V shunted rats

Organ wet weights, pleural effusion, and ascites. As shown in Table 2, the wet weights of the right and left ventricles and the lungs in the A-V shunted rats were significantly higher than in the control rats. In addition, pleural effusion and ascites were apparent in the A-V shunted rats.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Organ wet weights, pleural effusion, and ascites in the control and the A-V shunted rats

Evaluation of baseline SNA. As shown in Fig. 2A, 24-h urinary epinephrine and norepinephrine excretions were significantly greater in the A-V shunted rats than in the control rats (P < 0.01, P < 0.0001 vs. control, respectively). The baseline RSNA, which was evaluated by lowering the arterial pressure with an intravenous infusion of sodium nitroprusside, was significantly higher in the A-V shunted rats than in the control rats (P < 0.01, Fig. 2B). The actual RSNA levels before and after sodium nitroprusside were 61 ± 9 and 156 ± 18 spikes/s, respectively, in the control rats and 230 ± 40 (P < 0.01 vs. control) and 273 ± 39 spikes/s, respectively, in the A-V shunted rats. The changes in MAP did not differ between the two groups (-36 ± 7 vs. -38 ± 9 mmHg, respectively).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   A: 24-h urinary epinephrine (E) and norepinephrine (NE) excretions in the aortocaval (A-V) shunted rats and the sham control rats. B: baseline renal sympathetic nerve activity (RSNA) evaluated by decreasing arterial pressure with an intravenous infusion of sodium nitroprusside (see METHODS) in the A-V shunted rats and the sham control rats. Values are means ± SE. **P < 0.01, dagger P < 0.0001 vs. values with control rats.

Effects of microinjection of CV11974 or PD123319 into the NTS on arterial pressure, heart rate, and RSNA. The microinjection of L-glutamate into the NTS produced similar decreases in MAP (-15 ± 3 vs. -13 ± 3 mmHg, control vs. A-V shunted rats) and RSNA (-66 ± 14% vs. -65 ± 12%, control vs. A-V shunted rats) in the two groups.

The effects of microinjection of the AT1-receptor antagonist CV11974 into the NTS on arterial pressure, heart rate, and RSNA are shown in Figs. 3 and 4. The baseline values of these parameters are shown in Table 3. Microinjection of CV11974 into the NTS decreased arterial pressure and RSNA in both groups in a dose-dependent manner. The magnitudes of the decrease in MAP, heart rate, and RSNA were greater in the A-V shunted rats than in the control rats (Fig. 4). No changes in these parameters were observed after microinjection of either PD123319 or vehicle solution into the NTS.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3.   An original recording from an A-V shunted rat showing the responses of arterial pressure, heart rate, and integrated RSNA to a microinjection of CV11974 into the NTS.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Grouped data of responses of changes (Delta ) in mean arterial pressure (MAP; A), heart rate (HR; B), and RSNA (C) to a microinjection of CV11974 into the NTS in the control rats and the A-V shunted rats. Values are means ± SE. *P < 0.05, **P < 0.01 vs. values with control rats. dagger P < 0.05 vs. values with 50 pmol.


                              
View this table:
[in this window]
[in a new window]
  		Table 3.   The basal values before microinjection of CV11974

Effects of microinjection of CV11974 into the area postrema on arterial pressure, heart rate, and RSNA. Microinjection of CV11974 (500 pmol) into the area postrema did not alter arterial pressure (-0.6 vs. -2.4 mmHg), heart rate (-0.8 ± 0.8 vs. -1.0 ± 0.6 beats/min), and RSNA (0% vs. 2 ± 1%) in either group (control rats vs. A-V shunt rats, n = 5 for each).

ACE-receptor mRNA level. The expression of ACE-receptor mRNA in the lower brain stem was determined by Northern blot analysis, as shown in Fig. 5. Densitometric analysis demonstrated that the ratio of ACE mRNA to GAPDH mRNA was significantly higher in the A-V shunted rats than in the control rats (P < 0.05).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5.   Angiotensin-converting enzyme (ACE) expression in the rat medulla. A: autoradiograms of Northern blot analysis for ACE and GAPDH expression. B: densitmetric analysis of amount of ACE mRNA normalized against GAPDH mRNA in each experiment (n = 4). Values are means ± SE. *P < 0.05 vs. control group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A major finding in this study is that blocking the AT1 receptors, but not the AT2 receptors, in the NTS reduces arterial pressure, heart rate, and RSNA in rats with an A-V shunt. This suggests that activation of the renin-angiotensin system in the NTS via AT1 receptors contributes to an enhanced SNA in high output HF by an A-V shunt. Furthermore, we found an increase in ACE mRNA levels in the brain stem of rats with an A-V shunt, further supporting the hypothesis of activation of the renin-angiotensin system in the brain stem in this model.

First, we considered the possibility that the greater inhibition of arterial pressure, heart rate, and RSNA in response to the microinjection of CV11974 was due to the increased baseline vasoconstriction in A-V shunt model. This explanation seems unlikely, because a prior injection of L-glutamate produced similar depressor responses in the two groups. The L-glutamate injection, through the observed cardiovascular response to L-glutamate, was also important in the verification of the placement of the injection pipette in the dorsal medial NTS before injection of the AT1- or AT2-receptor antagonist. In addition, the vehicle solution was observed to have no effect on arterial pressure, heart rate, and RSNA, which excludes the possibility of nonspecific effects of the solution.

The A-V shunt model. In our model, high-output HF was produced by a volume overload through an arteriovenous fistula. This model has been used often as a model of HF (1, 6, 15, 17, 23, 45). However, this is an example of circulatory overload, which, in turn, leads to high-output HF (1, 6, 15, 17, 23, 45). Therefore, this model of HF is not consistent with that usually understood in the clinical context with a low cardiac output such as that in dilated cardiomyopathy or ischemic cardiomyopathy. In rats, a myocardial infarction through a coronary ligation is used as a low cardiac output model of HF. However, the mortality of that model is high, and the infarct size varies after a coronary ligation. On the other hand, a genetic model for dilated cardiomyopathy is not available in rats. The A-V fistula preparation of high-output HF is characterized by a circulatory overload. This model will eventually develop signs of ventricular failure with further increases in LVEDP (20). Furthermore, we induced a constriction of a portion of the aorta that was distal to the shunt portion. The addition of the aortic constriction produced HF that was more severe than in the usual arteriovenous shunt model demonstrated by other investigators (20). Our results clearly demonstrated an increased LVEDP, decreased LV dP/dt, enlarged LV end-diastolic and end-systolic diameters, and reduced ejection fraction. A postmortem examination also clearly demonstrated that the wet weights of the right and left ventricles and of the lungs were greater in the A-V shunted rats than in the control rats. In addition, pleural effusion and ascites were evident in the A-V shunted rats. More importantly, our model demonstrated neurohumoral activation, which is now considered an important target in the treatment of HF (32, 34). These results are consistent with those of previous studies (6, 11, 31, 32, 34). Sympathetic nervous system activation was demonstrated in two ways in our model. First, we found an elevated urinary norepinephrine excretion in our A-V shunt model, and second, we evaluated the baseline RSNA by reducing the arterial pressure with an intravenous infusion of sodium nitroprusside at the point determined to be the maximal RSNA. Our results are consistent with those obtained by Flaim et al. (15).

Activation of the renin-angiotensin system in the NTS in HF. The results of our study demonstrate that the renin-angiotensin system is activated in the NTS by high-output HF. We focused our efforts on the NTS, because this region is known to play an important role in central cardiovascular regulation and to process information from arterial and cardiopulmonary baroreceptors as well as chemoreceptors (3, 10). In addition, the NTS receives inputs from higher centers such as the hypothalamus (10). The primary neurotransmitter in the NTS is believed to be L-glutamate (3, 10). Moreover, ANG II is considered an important neuromodulator in the NTS as well as in other areas of the brain (3, 47); however, the roles played by ANG II in the NTS are not fully understood. Microinjection of ANG II into the NTS has been reported to yield not only pressor, but also depressor effects (16, 36, 42). Microinjection of low doses of ANG II into the NTS in the rat produced hypotension and bradycardia that mimicked stimulation of the baroreceptor reflex (16). In contrast, high doses of ANG II microinjected into the NTS increased arterial pressure (8, 42). The exact mechanisms by which ANG II produces pressor or depressor responses in the NTS are unknown. The activation of different receptor subtypes and affinities may be due to opposite effects of this peptide (3, 16). Alternatively, there may be interactions with primary or other neurotransmitter systems (42). In this case, qualitatively different responses would be possible through different states of activation of other participating systems. On the other hand, microinjection of a nonselective ANG II antagonist, such as [Sar1,Thr8]ANG II or [Sar1,Ala8]ANG II, into the NTS has been shown to facilitate arterial baroreceptor reflex control of heart rate (7, 36), suggesting that endogenous angiotensin reduces arterial baroreflex control. Furthermore, microinjection of the selective AT1-receptor antagonist CV11974 into the NTS facilitates baroreflex control of RSNA (33). Consistent with the results of our study, microinjection of an AT1-receptor antagonist, such as losartan or CV11974, did not alter baseline arterial pressure, heart rate, and/or RSNA in normotensive rats (16, 33). Matsumura et al. (33) also found no effect of microinjection of CV11974 into the NTS on baseline arterial pressure, heart rate, or RSNA in normotensive as well as in spontaneously hypertensive rats. Interestingly, we recently found that microinjection of CV11974 into the NTS produced dose-dependent decreases in arterial pressure, heart rate, and RSNA in hypertensive rats with chronic nitric oxide synthase inhibition (13). These findings are similar to the results of the present study. In our experiments, because the reflex effects on arterial pressure and RSNA unmask the effects of unilateral injection of the drug into the NTS, we performed microinjection of an AT1-receptor antagonist into the NTS after baroreceptor denervation to rule out secondary effects of this drug, because the reflex effects on arterial pressure and RSNA unmask the effects of unilateral injection of the drug into the NTS.

In addition to the microinjection experiments, we measured ACE-receptor mRNA levels in the lower brain stem and found that ACE mRNA levels were greater in the A-V shunted rats than in the sham control rats. Although these results suggest that ANG II in the lower brain stem may be increased in our A-V shunt model, the results of our study do not demonstrate this parameter directly, because we did not measure ANG II concentration in the NTS due to technical problems. Another explanation is that the number of AT1 receptors may be increased in our model. All components of the renin-angiotensin system exist independently in the brain as well as in various other peripheral organs and tissues (47). The brain is one of the major sites of ACE mRNA expression (46), and the NTS has been demonstrated to have few AT2 receptors (2, 4, 48). Thus it is possible that activation of the renin-angiotensin system occurs in the brain as well as peripheral organs and tissues in the A-V shunted rats via the AT1 receptors.

The brain renin-angiotensin system and the sympathetic nervous system in HF. Recent studies have demonstrated that the brain renin-angiotensin system is activated in HF via AT1 receptors and contributes to an enhanced SNA in HF. DiBona et al. (11) have shown that the intracerebroventricular administration of losartan to conscious rats with myocardial infarction decreases RSNA and increases arterial baroreflex gain. These results suggest that ANG II in the brain contributes to both the increased basal level of RSNA and the attenuated arterial baroreflex control of RSNA in HF. However, there are many sites of ANG II binding in the brain stem, including the NTS, the area postrema, and the rostral and caudal regions of the VLM (47). In the NTS, ANG II injection decreases arterial baroreflex gain, whereas a [Sar1,Thr8]ANG II injection increases arterial baroreflex gain (7). Zhang et al. (51) have shown that the chronic increase in sympathetoexcitation, decrease in sympathoinhibition, and desensitized baroreflex function in HF may depend on the brain renin-angiotensin system, because these changes can be normalized by a chronic central AT1-receptor blockade with losartan. In the same laboratory, Leenen et al. (26) demonstrated that increased brain levels of ouabain activate the brain renin-angiotensin system in HF. However, the precise mechanisms and sites by which the brain renin-angiotensin system is activated in HF cannot be determined based on the results of these studies. Our results indicate the importance of the NTS in the activation of the renin-angiotensin system and the subsequent increase in RSNA in high-output HF due to an A-V shunt.

Areas in the brain other than the NTS may play a role in activation of the sympathetic nervous system and renin-angiotensin system. Recent studies using immunohistochemical detection of Fra-like immunoreactivity as a marker of long-term neuronal activation have demonstrated that the paraventricular nucleus, supraoptic nucleus, and locus ceruleus as well as the NTS have significant increases in numbers of Fra-positive neurons in rats with myocardial infarction (44). Further studies will be needed to examine whether these areas also exhibit activation of the renin-angiotensin system in HF leading to an enhanced SNA. Although AT1 receptors are rich in the area postrema, we found no effect of microinjection of CV11974 into the area postrema. The significance and exlanation for this finding is unclear.

Limitations of this study. As mentioned above, our model is not a low cardiac output model of HF. Although the A-V shunt model produces high-output HF and neurohumoral activation (15, 45), further studies will be needed to determine whether activation of the renin-angiotensin system in the NTS occurs in a low cardiac output model of HF.

We used sodium nitroprusside to determine the maximal level of RSNA as one of the markers of the increased SNA in this model. Because baroreflex function is abnormal in HF, determining the maximal level of RSNA by this method is difficult. However, the reduction of arterial pressure by ~40 mmHg is known to produce maximal increases in SNA that eventually reach a plateau (43). The reduction of arterial pressure by infusion of sodium nitroprusside did not differ between the A-V shunted rats and the control rats. Furthermore, we measured the urinary catecholamine excretion as another index of increased SNA in the A-V shunt model. Renal function may be impaired in rats with an A-V shunt, and normalization by urinary creatinine excretion would be an improved method. However, we observed clear differences without such normalization.

We evaluated multifiber recordings of RSNA between rats by counting spikes per second, which might underestimate the level of RSNA (22). Therefore, we normalized RSNA, as shown in Fig. 2B, although we observed a significantly increased baseline RSNA. Clear differences were still observed between the two groups. Thus we believe that endogenous ANG II, via AT1 receptors, increases RSNA in our model.

In summary, our results strongly suggest that endogenous ANG II in the NTS contributes to the increased sympathetic drive in high-output HF generated by an A-V shunt via AT1 receptors but not via AT2 receptors.

Perspectives

Recently, Liu and Zucker (30) proposed the hypothesis that both a loss of nitric oxide and an increase in ANG II are necessary for sustained increases in SNA in HF. Nitric oxide has been shown to play an important role in the NTS in controlling the sympathetic nervous system (19). Thus the nitric oxide system may be altered in HF (40, 49, 50) and contribute to enhanced SNA as well as activation of the renin-angiotensin system. In addition, recent clinical studies have demonstrated the beneficial effects of ACE inhibitors and AT1-receptor antagonists in patients with HF (32, 34). Some evidence suggests that, after oral administration, AT1-receptor antagonists such as losartan cross the blood-brain barrier and affect the brain renin-angiotensin system (28). Development of a new AT1-receptor antagonist, which has more affinity for the brain, may be desirable for the treatment of patients with chronic HF.


    ACKNOWLEDGEMENTS

This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (C11670689).


    FOOTNOTES

The results of this study were presented in part at the 71st Scientific Sessions of the American Heart Association, Dallas, TX, November 8-11, 1998 (Circulation 98: I-211, 1998).

Address for reprint requests and other correspondence: Y. Hirooka, Dept. of Cardiovascular Medicine, Cardiovascular Science, Graduate School of Medical Sciences, Kyushu Univ., 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (E-mail: hyoshi{at}cardiol.med.kyushu-u.ac.jp).

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.

Received 7 August 2000; accepted in final form 7 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abassi, ZA, Gurbanov K, Mulroney SE, Potlog C, Opgenorth TJ, Hoffman A, Haramati A, and Winaver J. Impaired nitric oxide-mediated renal vasodilation in rats with experimental heart failure. Circulation 96: 3655-3664, 1997[Abstract/Free Full Text].

2.   Allen, AM, Moeller I, Jenkins TA, Zhuo J, Aldred GP, Chai SY, and Mendelsohn FAO Angiotensin receptors in the nervous system. Brain Res Bull 47: 17-28, 1998[ISI][Medline].

3.   Andresen, MC. Nucleus tractus solitarius---gateway to neural circulatory control. Annu Rev Physiol 56: 93-116, 1994[ISI][Medline].

4.   Barnes, KL, McQueeney AJ, and Ferrario CM. Receptor subtype that mediates the neuronal effects of angiotensin II in the rat dorsal medulla. Brain Res Bull 31: 195-200, 1993[ISI][Medline].

5.   Brändle, M, Patel KP, Wang W, and Zucker IH. Hemodynamic and norepinephrine responses to pacing-induced heart failure in conscious sinoaortic-denervated dogs. J Appl Physiol 81: 1855-1862, 1996[Abstract/Free Full Text].

6.   Brodski, S, Gurbanov K, Abassi Z, Hoffman A, Ruffodo RR, Jr, Feuerstein GZ, and Winaver J. Effects of eprosartan on renal function and cardiac hypertrophy in rats with experimental heart failure. Hypertension 32: 746-752, 1998[Abstract/Free Full Text].

7.   Campagnole-Santos, MJ, Diz DI, and Ferrario CM. Baroreceptor reflex modulation by angiotensin II at the nucleus tractus solitarii. Hypertension 11, Suppl1: I167-I171, 1988.

8.   Casto, R, and Phillips MI. Mechanism of pressor effects by angiotensin in the nucleus tractus solitarius of rats. Am J Physiol Regulatory Integrative Comp Physiol 247: R575-R581, 1984.

9.   Chevillard, C, and Saavedra JM. Distribution of angiotensin converting enzyme activity in specific areas of the rat brain stem. J Neurochem 38: 281-284, 1982[ISI][Medline].

10.   Dampney, RAL Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74: 323-364, 1994[Free Full Text].

11.   DiBona, GF, Jones SY, and Brooks VL. ANG II receptor blockade and arterial baroreflex regulation of renal nerve activity in cardiac failure. Am J Physiol Regulatory Integrative Comp Physiol 269: R1189-R1196, 1995[Abstract/Free Full Text].

12.   Dzau, VJ, Ingelfinger J, Pratt RE, and Ellison KE. Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains. Hypertension 8: 544-548, 1986[Abstract/Free Full Text].

13.   Eshima, K, Hirooka Y, Shigematsu H, Matsuo I, Koike G, Sakai K, and Takeshita A. Angiotensin in the nucleus tractus solitarii contributes to neurogenic hypertension caused by chronic nitric oxide synthase inhibition. Hypertension 36: 259-263, 2000[Abstract/Free Full Text].

14.   Feng, QP, Carlsson S, Thoren P, and Hender T. Characteristics of renal sympathetic nerve activity in experimental congestive heart failure in the rat. Acta Physiol Scand 150: 259-266, 1994[ISI][Medline].

15.   Flaim, SF, Minteer WJ, Nellis SH, and Clark DP. Chronic arteriovenous shunt: evaluation of a model for heart failure in rat. Am J Physiol Heart Circ Physiol 236: H698-H704, 1979[Abstract/Free Full Text].

16.   Fow, JE, Averill DB, and Barnes KL. Mechanisms of angiotensin-induced hypotension and bradycardia in the medial solitary tract nucleus. Am J Physiol Heart Circ Physiol 267: H259-H266, 1994[Abstract/Free Full Text].

17.   Garcia, R, and Diebold S. Simple, and rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res 24: 430-432, 1990[Abstract/Free Full Text].

18.   Gehlert, DR, Gackenheimer SL, and Schober DA. Autoradiographic localization of subtypes of angiotensin II antagonist binding in the brain. Neuroscience 44: 501-514, 1991[ISI][Medline].

19.   Harada, S, Tokunaga S, Momohara M, Masaki H, Tagawa T, Imaizumi T, and Takeshita A. Inhibition of nitric oxide formation in the nucleus tractus solitarius increases renal sympathetic nerve activity in rabbits. Circ Res 72: 511-516, 1993[Abstract/Free Full Text].

20.   Hirano, S, Imamura T, Matsuo T, Ishiyama Y, Kato J, Kitamura K, Koiwaya Y, and Eto T. Differential responses of circulating and tissue adrenomedullin and gene expression to volume overload. J Card Fail 6: 120-129, 2000[ISI][Medline].

21.   Hirooka, Y, Polson JW, and Dampney RAL Pressor and sympathoexcitatory effects of nitric oxide in the rostral ventrolateral medulla. J Hypertens 14: 1317-1324, 1996[ISI][Medline].

22.   Hopp, FA, Seagard JL, and Kampine JP. Comparison of four methods of averaging nerve activity. Am J Physiol Regulatory Integrative Comp Physiol 251: R700-R711, 1986[Abstract/Free Full Text].

23.   Huang, M, LeBlanc MH, and Hester R. Evaluation of the needle technique for producing an arteriovanous fistula. J Appl Physiol 77: 2907-2911, 1994[Abstract/Free Full Text].

24.   Koike, G, Kriger JE, Jacob HJ, Mukoyama M, Pratt RE, and Dzau VJ. Angiotensin converting enzyme and genetic hypertension: cloning of rat cDNAs and characterization of the enzyme. Biochem Biophys Res Commun 198: 380-386, 1994[ISI][Medline].

25.   Krassioulov, AV, and Weaver LC. Connections between the pontine reticular formation and rostral ventrolateral medulla. Am J Physiol Heart Circ Physiol 265: H1386-H1392, 1993[Abstract/Free Full Text].

26.   Leenen, FHH, Yuan B, and Huang BS. Brain "ouabain" and angiotensin II contribute to cardiac dysfunction after myocardial infarction. Am J Physiol Heart Circ Physiol 277: H1786-H1792, 1999[Abstract/Free Full Text].

27.   Levett, JM, Marinelli CC, Lund DD, Pardini BJ, Nader S, Scott BD, Augelli NV, Kerber RE, and Schmid PD, Jr. Effects of beta -blockade on neurohumoral responses and neurochemical markers in pacing-induced heart failure. Am J Physiol Heart Circ Physiol 266: H468-H475, 1994[Abstract/Free Full Text].

28.   Li, Z, Bains JS, and Ferguson AV. Functional evidence that the angiotensin losartan crosses the blood-brain barrier in the rat. Brain Res Bull 30: 33-39, 1993[ISI][Medline].

29.   Lind, RW, Swanson LW, and Ganten D. Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. Neuroendocrinology 40: 2-24, 1985[ISI][Medline].

30.   Liu, J-L, and Zucker IH. Regulation of sympathetic nerve activity in heart failure: a role for nitric oxide and angiotensin II. Circ Res 84: 417-423, 1999[Abstract/Free Full Text].

31.   Lohmeier, TE, Mizelle HL, Reinhart GA, and Montani J-P. Influence of angiotensin on the early progression of heart failure. Am J Physiol Regulatory Integrative Comp Physiol 278: R74-R86, 2000[Abstract/Free Full Text].

32.   Mark, AL. Sympathetic dysregulation in heart failure: mechanisms and therapy. Clin Cardiol 18, SupplI: I-3-I-8, 1995.

33.   Matsumura, K, Averill DB, and Ferrario CM. Angiotensin II acts at AT1 receptors in the nucleus of the solitary tract to attenuate the baroreceptor reflex. Am J Physiol Regulatory Integrative Comp Physiol 275: R1611-R1619, 1998[Abstract/Free Full Text].

34.   Middlekauff, HR, and Mark AL. The treatment of heart failure: the role of neurohumoral activation. Intern Med 37: 112-122, 1998[ISI][Medline].

35.   Mungall, BA, Shinkel TA, and Sernia C. Immunocytochemical localization of angiotensinogen in the fetal and neonatal rat brain. Neuroscience 67: 505-524, 1995[ISI][Medline].

36.   Mosqueda-Garcia, R, Tseng C-J, Appalsamy M, and Robertson D. Cardiovascular effects of microinjection of angiotensin II in the brainstem of renal hypertensive rats. J Pharmacol Exp Ther 255: 374-381, 1990[Abstract/Free Full Text].

37.   Murakami, H, Liu J-L, and Zucker IH. Blockade of AT1 receptors enhances baroreflex control of heart rate in conscious rabbits with heart failure. Am J Physiol Regulatory Integrative Comp Physiol 271: R303-R309, 1996[Abstract/Free Full Text].

38.   Murakami, H, Liu J-L, and Zucker IH. Angiotensin II enhances baroreflex control of sympathetic outflow in heart failure. Hypertension 29: 564-569, 1997[Abstract/Free Full Text].

39.  Numaguchi K, Egashira K, Takemoto M, Kadokami T, Shimokawa H, Sueishi K, and Takeshita A. Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats. Hypertension 26: 957-962.

40.   Patel, KP, Zhang K, Zucker IH, and Krukoff TL. Decreased gene expression of neuronal nitric oxide synthase in hypothalamus and brainstem of rats in heart failure. Brain Res 734: 109-115, 1996[ISI][Medline].

41.   Rector, TS, Olivari MT, Levine TB, Francis GS, and Cohn JN. Predicting survival for an individual with congestive heart failure using the plasma norepinephrine concentration. Am Heart J 114: 148-152, 1987[ISI][Medline].

42.   Rettig, R, Healy DP, and Printz MP. Cardiovascular effects of microinjections of angiotensin II into the nucleus tractus solitarii. Brain Res 364: 233-240, 1986[ISI][Medline].

43.   Takishita, S, and Ferrario CM. Altered control of cardiovascular function in sodium-depleted dogs. Hypertension 4, SupplII: 175-182, 1982[Abstract/Free Full Text].

44.   Vahid-Ansari, F, and Leenen FHH Pattern of neuronal activation in rats with CHF after myocardial infarction. Am J Physiol Heart Circ Physiol 275: H2140-H2146, 1998[Abstract/Free Full Text].

45.   Villarreal, D, Freeman RH, and Johnson RA. Neurohumoral modulators and sodium balance in experimental heart failure. Am J Physiol Heart Circ Physiol 264: H1187-H1193, 1993[Abstract/Free Full Text].

46.   Whiting, P, Nava S, Mozley L, Eastham H, and Poat J. Expression of angiotensin converting enzyme mRNA in rat brain. Mol Brain Res 11: 93-96, 1991[Medline].

47.   Wright, JW, and Harding JW. Regulatory role of brain angiotensins in the control of physiological and behavioral responses. Brain Res Rev 17: 227-262, 1992[Medline].

48.   Wright, JW, and Harding JW. Brain angiotensin receptor subtypes in the control of physiological and behavioral responses. Neurosci Biobehav Rev 18: 21-53, 1994[ISI][Medline].

49.   Zhang, K, and Patel KP. Effects of nitric oxide within the paraventricular nucleus on renal sympathetic nerve discharge: role of GABA. Am J Physiol Regulatory Integrative Comp Physiol 275: R728-R734, 1998[Abstract/Free Full Text].

50.   Zhang, K, Zucker IH, and Patel KP. Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure. Brain Res 786: 219-225, 1998[ISI][Medline].

51.   Zhang, W, Huang BS, and Leenen FHH Brain renin-angiotensin system and sympathetic hyperactivity in rats after myocardial infarction. Am J Physiol Heart Circ Physiol 276: H1608-H1615, 1999[Abstract/Free Full Text].

52.   Zucker, IH, Wang W, Brändle M, Schultz HD, and Patel KP. Neural regulation of sympathetic nerve activity in heart failure. Prog Cardiovasc Dis 37: 397-414, 1995[ISI][Medline].

Am J Physiol Regul Integr Comp Physiol 280(6):R1665-R1673
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W.-Z. Wang, L. Gao, H.-J. Wang, I. H. Zucker, and W. Wang
Interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the NTS AT1 receptors in heart failure
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1216 - H1226.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
B. Bishara, H. Shiekh, T. Karram, I. Rubinstein, Z. S. Azzam, N. Abu-Saleh, S. Nitecki, J. Winaver, A. Hoffman, and Z. A. Abassi
Effects of Novel Vasopressin Receptor Antagonists on Renal Function and Cardiac Hypertrophy in Rats with Experimental Congestive Heart Failure
J. Pharmacol. Exp. Ther., August 1, 2008; 326(2): 414 - 422.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
W.-Z. Wang, L. Gao, Y.-X. Pan, I. H. Zucker, and W. Wang
AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1137 - R1145.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Sakima, D. B. Averill, S. O. Kasper, L. Jackson, D. Ganten, C. M. Ferrario, P. E. Gallagher, and D. I. Diz
Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen
Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1412 - H1419.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Sherrod, D. R. Davis, X. Zhou, M. D. Cassell, and C. D. Sigmund
Glial-specific ablation of angiotensinogen lowers arterial pressure in renin and angiotensinogen transgenic mice
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1763 - R1769.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Sakai, Y. Hirooka, H. Shigematsu, T. Kishi, K. Ito, H. Shimokawa, A. Takeshita, and K. Sunagawa
Overexpression of eNOS in brain stem reduces enhanced sympathetic drive in mice with myocardial infarction
Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H2159 - H2166.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
P. B. Persson
The kidney and hypertension
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2003; 284(5): R1176 - R1178.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
T. E. Lohmeier
Neurohumoral regulation of arterial pressure in hemorrhage and heart failure
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R810 - R814.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
H. M. Stauss
Baroreceptor reflex function
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2002; 283(2): R284 - R286.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
O. Skott
Renin
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R937 - R939.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar