Copyright © 2006, European Society of Cardiology
Angiotensin II enhances carotid body chemoreflex control of sympathetic outflow in chronic heart failure rabbits
aDepartment of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850, USA
bDepartment of Pathophysiology, Hebei Academy of Medical Science, Shijiazhuang, Hebei 050021, People's Republic of China
cDivision of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, SD 57069-2390, USA
* Corresponding author. Tel.: +1 402 559 7167; fax: +1 402 559 4438. Email address: hschultz{at}unmc.edu
Received 26 January 2006; revised 14 March 2006; accepted 20 March 2006
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objectives We investigated whether Angiotensin II (Ang II) modulates peripheral chemoreflex function through carotid body (CB) chemoreceptors in chronic heart failure (CHF).
Methods We measured renal sympathetic nerve activity (RSNA) in response to graded hypoxia before and after intravenous administration of Ang II (20ng/kg/min, i.v. 30min) or AT1 receptor antagonist (L-158,809, 0.33mg/kg, i.v.) in conscious sham and pacing-induced CHF rabbits. We also investigated the effects of Ang II (100pM) and L-158,809 (1µM) on CB chemoreceptor activity in vascularly isolated–perfused CB preparations from sham and CHF rabbits.
Results Ang II enhanced hypoxia-induced RSNA increases in sham rabbits but not in CHF rabbits. Conversely, L-158,809 attenuated hypoxia-induced responses in RSNA in CHF rabbits but not in sham rabbits. Using RT-PCR, Western blotting, and immunocytochemistry, we found that the mRNA and protein expression of AT1 receptor in the CB from CHF rabbits were greater than that in sham rabbits. CB chemoreceptor afferent activity during normoxia and graded hypoxia was increased in CHF rabbits compared with sham rabbits. Ang II increased the response of CB chemoreceptors to hypoxia in sham rabbits but not CHF rabbits. L-158,809 decreased CB chemoreceptor responses to hypoxia in CHF rabbits but not in sham rabbits.
Conclusions These results indicate that elevation of Ang II and concomitant upregulation of AT1 receptor in the CB contribute to the increased CB chemoreceptor activity and enhanced peripheral chemoreflex function in CHF.
KEYWORDS Angiotensin; Autonomic nervous system; Chemoreflex; Heart failure; Hypoxia
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Angiotensin II (Ang II) is a physiologically active component of the renin–Ang system, which plays an important role in the maintenance of blood pressure and fluid homeostasis [1]. It is well documented that systemic and tissue Ang II levels are increased during chronic heart failure (CHF) in patients and animal models [2–5]. Ang II has been confirmed to increase carotid body (CB) chemoreceptor activity via the AT1 receptor [6]. Furthermore, a locally generated Ang II system is operational [7], and chronic hypoxia upregulates the expression of the AT1 receptors in the rat CB [8].
It is known that an enhancement of peripheral chemoreflex sensitivity occurs in pacing-induced CHF rabbits [9]. This enhanced sensitivity of the peripheral chemoreflex contributes to sympathetic activation in the CHF state since inhibition of peripheral chemoreceptor activity is shown to decrease resting renal sympathetic nerve activity (RSNA) [9]. An augmented afferent input from the CB chemoreceptors is involved in the enhancement of peripheral chemoreflex function in CHF rabbits [10]. In addition, plasma Ang II concentration is elevated in the CHF rabbits [2].
Because Ang II influences CB chemoreceptor activity [6] and Ang II concentration is elevated in the CHF rabbits [2], we sought to determine if Ang II contributes to enhanced peripheral chemoreflex function in CHF. We first measured RSNA and minute ventilation (VE) in response to graded hypoxia before and after intravenous administration of Ang II or an AT1 receptor antagonist (L-158,809) in sham and CHF rabbits. Secondly, we measured mRNA and protein expression of the AT1 receptor in the CB from sham and CHF rabbits in order to determine if the enhanced peripheral chemoreflex function is concomitant with the over-expression of AT1 receptor in the CB from CHF rabbits. Thirdly, we investigated the effects of Ang II and L-158,809 on the CB chemoreceptor activity in sham and CHF rabbits
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2. Methods |
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2.1 Pacemaker implant and production of CHF
All experiments were carried out on male New Zealand White rabbits weighing 2.5–3.5kg. Experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the American Physiological Society's Guides for the Care and Use of Laboratory Animals. Rabbits were assigned to sham-operated and CHF groups. They were housed in individual cages under controlled temperature and humidity and a 12:12-h dark–light cycle, and fed standard rabbit chow with water available ad libitum.
Rabbits were anesthetized with a cocktail consisting of 1.2mg acepromazine, 5.9mg xylazine and 58.8mg ketamine, given as an i.m. injection. Using sterile technique, a left thoracotomy was performed as previously described [9]. Briefly, a pin electrode was attached to the left ventricle for pacing. Two sonomicrometer crystals (Sonometrics Corp., London, ON, Canada) were attached to opposing walls of the lateral left ventricle for measuring external diameter. The chest was closed. Rabbits were placed on an antibiotic regimen consisting of 5mg/kg Baytril i.m. for 5days. After 2weeks, baseline left-ventricular end-systolic and end-diastolic external diameter (D), fractional shortening, and shortening velocity (dD/dtmax) were measured by sonomicrometry (Triton Technology Inc., San Diego, CA, USA). Sonograms and experimental procedures were performed with the pacemaker turned off for at least 60min before the recordings were started. The pacing was started at 320bpm, held for 7days, and then the rate was gradually increased to 380bpm, with an increment of 20bpm each week. Rabbits with >40% reductions in dD/dtmax and shortening fraction are considered in CHF (generally after 3–4weeks). Sham-operated animals underwent a similar period of sonographic measurements with the pacemaker turned off. Any rabbit exhibiting abnormal arterial blood gases (PaO2<85mm Hg; 45mm Hg<PaCO2<30mm Hg) were excluded from study [9].
2.2 Immunofluorescence for AT1 receptor and tyrosine hydroxylase (TH) detection
Both CBs in each rabbit were rapidly removed and postfixed in 4% paraformaldehyde for 12h at 4°C, followed by soaking the CBs in 30% of sucrose for 12h at 4°C for cryostat protection. The CB was cut into 10µm-thick sections. The CB sections were mounted on precoated glass slides for immunofluorescence for AT1 receptor and TH detection. CB sections on the glass slide were incubated with 10% of normal donkey serum for 1h followed by incubation with primary anti-AT1 receptor and anti-TH antibodies (Santa Cruz, CA, USA) overnight at 4°C. Then the sections were incubated with appropriate secondary antibody (Molecular Probe, Carisbad, CA, USA) for 60min at room temperature. At last, slides were observed under a Leica fluorescent microscope with appropriate excitation/emission filters. Pictures were captured by a digital camera system. No staining was seen when the procedure described above was used but PBS was used instead of the primary antibody.
2.3 Semi-quantitative RT-PCR for AT1 receptor
CBs were rapidly removed and immediately frozen in dry ice and stored at – 80°C until analyzed. The detailed procedures of RT-PCR for AT1 receptor mRNA have been reported [11]. Briefly total RNA was isolated by means of the RNeasy Mini Kit Total RNA Isolation System (QIAGEN Inc., Valencia, CA, USA), and then cDNA was synthesized by means of M-MLV Reverse Transcriptase (Invitrogen Life technologies, Carisbad, CA, USA), according to the manufacturer's instructions. PCR amplification was performed by means of a PTC-100 Programmable Thermal Controller (MJ Research, Inc., Watertown, MA, USA) as follows: 1 cycle at 95°C for 15min, followed by 35 cycles of 94°C for 45s, 55°C for 45s, and 72°C for 1min. The primer pairs were based on the cDNA sequences of rabbit AT1 receptor with β-actin as an internal control. The primer pairs were 5'-TTTGGGAACAGCTTGGC-GGT-3' and 5'-GCCAGCC AGCAGCCAAATAA-3' for AT1 receptor and 5'-GATCGC-TGACCGTATGCAG-3' and 5'-GTCGT ACTCCTGCTTGGTG-3' for β-actin. The amplification products were visualized on 2% agarose gels by the use of ethidium bromide and sequenced so that their identity could be confirmed. The bands were analyzed using UVP BioImaging Systems (UVP Inc, Upland, CA, USA).
2.4 Western blot analysis for AT1 receptor
CBs were obtained in the same manner as described above. The protein was extracted with the lysing buffer (10mM PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS) plus protease inhibitor cocktail (100µl/ml). Following a centrifugation at 12,000g for 20min at 4 °C, the protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). The protein sample was mixed with loading buffer containing β-mercaptoethanol and heated at 100°C for 5min. Five µg of protein was loaded. Protein was fractionated in a 10% polyacrylamide gel along with molecular weight standards and transferred to PVDF membrane. The membrane was probed with a mouse anti-AT1 antibody (Santa Cruz, CA, USA) and a peroxidase-conjugated goat anti-mouse IgG. The signal was detected using enhanced chemiluminescence substrate (Pierce Chemical, Rockford, IL, USA) and the bands were analyzed using UVP BioImaging Systems. Protein loading was controlled by probing all Western blots with anti-β-tubulin antibody (Santa Cruz) and normalizing AT1 receptor protein intensity to that of β-tubulin.
2.5 Plasma and CB Ang II concentration
Plasma was prepared from arterial blood and tissue homogenate was prepared from CB samples. The plasma and CB Ang II concentrations were measured by Ang II 125I radioimmunoassay kit (Buhlmann Laboratories, Switzerland). The final Ang II concentration was counted by 1470 Automatic Gamma Counter (PerkinElmer, Shelton, CT, USA) and calculated with a standard curve generated for each experiment.
2.6 Renal sympathetic nerve activity (RSNA) and minute ventilation (VE)
RSNA recording electrodes were implanted as described previously [9]. At that time, arterial/venous catheters were inserted into the right carotid artery and jugular vein. RSNA recording was performed 3days after surgery. Changes in RSNA and VE in response to the stimulation of peripheral chemoreceptors were measured in sham and CHF rabbits in the conscious state as described previously [9]. RSNA was expressed as % maximum, and maximal RSNA was determined in each rabbit by an intravenous bolus injection of sodium nitroprusside (100µg/kg) [2]. VE was calculated by the equation (tidal volume x breathing rate/body weight) [9]. Peripheral chemoreceptors were stimulated preferentially by allowing the rabbits to breathe graded mixtures of hypoxic gas (3–5min) under isocapnic conditions. Because hypoxic stimulation of ventilation induces hyperventilatory hypocapnia, 2–4% CO2 was added to the hypoxic gases to maintain relatively constant PaCO2 during hyperventilation [9]. PaO2, PaCO2 and pH of arterial blood were measured by a blood gas analyzer (ABL5, Radiometer, Copenhagen, Denmark).
Peripheral chemoreflex function curves were analyzed by an equation described as RSNA or VE=a+b/(PaO2 – c) [9]. The parameter b is a slope coefficient and was used to compare the peripheral chemoreflex sensitivity among the groups.
2.7 CB chemoreceptor fiber activity
Single unit action potentials were recorded from CB chemoreceptor fibers in the carotid sinus nerve (CSN) as we have described previously [10]. Briefly, the left or right carotid sinus region was vascularly isolated and perfused with Krebs–Henseleit solution (in mM: 120 NaCl, 4.8 KCl, 2.0 CaCl2, 2.5MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 5.5 glucose; 10ml/min, T 37°C). Perfusate was bubbled with O2/CO2/N2 gas mixture to maintain PO2 at 100–110mm Hg, PCO2 at 30–35mm Hg, and pH at 7.4 as the normoxic condition. PO2, PCO2 and pH of the buffer solution perfusing the carotid sinuses were measured by gas- and ion-selective electrodes with 1201 chemical microsensor (Diamond General, Ann Arbor, MI, USA). Flow through the isolated sinus was set at 10ml/min at a perfusion pressure of 80mm Hg via a screw clamp on the effluent line, which was adjusted throughout the experiments to keep flow and pressure constant.
Autonomic innervation to the carotid sinus region was eliminated by stripping all visible neural connections among the carotid sinus, the superior cervical, and nodose ganglia. The CSN was exposed and transected near the petrosal ganglion to interrupt neural efferents to the CB.
The CSN was covered with mineral oil, and fine slips of nerve filaments were placed on a silver electrode. Impulses were amplified with a bandwidth of 100Hz–3 kHz (Grass P511, Grass Instrument, Quincy, MA, USA), displayed on an oscilloscope (2120 Oscilloscope, BK Precision, Taiwan), and fed into a rate meter (FHC, Brunswick, ME, USA) whose window discriminators were set to accept potentials of the particular amplitude. Bundles that had one, or at most two, easily distinguishable active fibers were used. Chemoreceptor afferents were identified by their sparse and irregular discharge at normoxia and by their response to hypoxia and NaCN.
2.8 Drugs
L-158,809 was a gift from Merck Co., NJ, USA. Other chemicals used in this study were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA.
2.9 Data analysis
All data are presented as means±SEM. Statistical significance was determined by student's paired t test for hemodynamic parameters, and student's unpaired t test for mRNA and protein expression of AT1 receptor. A two-way ANOVA, with a Bonferroni procedure for post hoc was used in comparisons of RSNA, VE, and single-unit activity of CB chemoreceptor afferent nerve. Statistical significance was accepted when p<0.05.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1 Induction of CHF
Rapid left-ventricular pacing induced CHF by the 3rd or 4th week of pacing, consistent with previous studies [9]. LV dD/dtmax and LV shortening fraction were reduced by 58.6±1.4% and 59.4±1.2%, respectively, from prepaced baselines (p<0.05, Table 1). There was no change in the LV dD/dtmax and LV shortening fraction over the course of 3–4weeks in sham rabbits (Table 1).
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3.2 Effects of Ang II and L-158,809 on MBP and HR in sham and CHF rabbits
Ang II infusion (20ng/kg/min, i.v., 30min) increased baseline MBP and decreased HR in sham rabbits (p<0.05) but had no significant effect on MBP and HR in CHF rabbits (Table 2). The AT1 receptor antagonist, L-158,809 (0.33mg/kg, i.v.), did not induce significant changes in MBP and HR in sham or CHF rabbits (Table 2). Exposure to graded levels of hypoxia did not change MBP and HR in sham and CHF rabbits (data not shown).
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3.3 Effect of Ang II on the peripheral chemoreflex in sham and CHF rabbits
Baseline RSNA and VE at normoxia in the CHF rabbits were elevated compared to those in sham rabbits (Fig. 1). Similarly, the isocapnic hypoxia-induced increases in RSNA and VE were significantly augmented in the CHF rabbits vs. sham rabbits (Fig. 1, Table 3).
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p<0.05 vs. control in sham group.
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In sham rabbits, intravenous infusion of Ang II (20ng/kg/min, i.v., 30min) increased MBP (Table 2), decreased the baseline RSNA (from 21.5±1.9% of max to 10.7±1.5% of max) and enhanced RSNA and VE responses to isocapnic hypoxia (Table 3). The decrease in baseline RSNA was likely due to the involvement of the baroreflex. Therefore, hydralazine (0.01 to 0.06mg/kg/min, i.v.) was infused simultaneously with Ang II to keep MBP at the control level [12] and to eliminate the effect of MBP on RSNA. In sham rabbits with hydralazine infusion, Ang II infusion did not affect baseline RSNA and VE at normoxia (Fig. 1), but it similarly enhanced the responses of RSNA and VE to isocapnic hypoxia (Fig. 1, Table 3). Pretreatment with L-158,809 (0.33mg/kg, i.v.) inhibited the effects of Ang II to increase MBP and to enhance RSNA and VE responses to hypoxia (Fig. 1).
Since Ang II infusion did not alter baseline MBP in CHF rabbits (Table 2), hydralazine infusion was not employed. In CHF rabbits, Ang II infusion (20ng/kg/min, i.v., 30min) neither altered baseline RSNA and VE (Fig. 1), nor altered responses of RSNA and VE to isocapnic hypoxia (Fig. 1, Table 3).
In sham rabbits, Ang II infusion increased plasma Ang II concentration from 12.8±3.7 to 56.6±4.2pM (n=7, p<0.05). The plasma Ang II level after infusion in sham rabbits was the same as the baseline level of plasma Ang II in CHF rabbits (49.6±6.6pM, n=7), and similar to that measured previously [2].
3.4 Effect of L-158,809 on the peripheral chemoreflex in sham and CHF rabbits
The AT1 receptor antagonist, L-158,809 (0.33mg/kg, i.v.), alone did not affect RSNA and VE either at normoxia (baseline) or in response to isocapnic hypoxia in sham rabbits. L-158,809 had no effect on baseline RSNA and VE in CHF rabbits (Fig. 2, Table 3). However, L-158,809 significantly attenuated the responses of RSNA and VE to isocapnic hypoxia in CHF rabbits (Fig. 2, Table 3).
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3.5 mRNA and protein expression of AT1 receptor in CBs from sham and CHF rabbits
Using a double-labeling technique, we found that distinct immunostaining of the AT1 receptor was predominantly localized to the cell clusters of CB glomus cells (Fig. 3), which was validated by the presence of TH as an immunohistochemical marker for CB glomus cells [13]. Immunostaining for AT1 receptor was increased in the CB glomus cells from CHF rabbits vs. sham rabbits (Fig. 3). Similarly, the mRNA and protein expression of the AT1 receptor were increased in CBs from CHF rabbits vs. sham rabbits (Fig. 4).
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3.6 Effects of Ang II and L-158,809 on CB chemoreceptor activity in sham and CHF rabbits
The baseline CB chemoreceptor activity at normoxia and its response to isocapnic hypoxia were enhanced in CHF rabbits vs. sham rabbits (Fig. 5), consistent with data from our previous study [10]. In sham rabbits, perfusion of the isolated sinus with Ang II (100pM) did not affect the baseline discharge at normoxic, but it increased the CB chemoreceptor response to hypoxia (Fig. 5). Simultaneous administration of L-158,809 inhibited the effect of Ang II to enhance CB chemoreceptor responses to hypoxia (Fig. 5). L-158,809 (1µM) alone had no effect on CB chemoreceptor activity at normoxia or hypoxia in sham animals (data not shown).
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In CHF rabbits, Ang II perfusion (100pM) did not change either the baseline CB chemoreceptor discharge or the chemoreceptor response to hypoxia. L-158,809 (1µM) alone tended to decrease baseline CB chemoreceptor activity at normoxia in CHF rabbits, but the effect was not significant. However, L-158,809 markedly decreased the chemoreceptor response to hypoxia in CHF rabbits (Fig. 5). The tissue Ang II concentration in the CBs from CHF rabbits was significantly elevated (35.6±1.4pg/mg protein, n=8), compared to that in sham rabbits (12.6±0.8pg/mg protein, n=8, p<0.05).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The present study demonstrates that elevated circulating Ang II augments hypoxia-induced increases in RSNA and VE in conscious sham rabbits but not in CHF rabbits. On the other hand, the AT1 receptor antagonist, L-158,809, blunts hypoxia-induced responses in RSNA and VE in CHF rabbits but not in sham rabbits. Similarly, Ang II administration to the CB enhances CB chemoreceptor responsiveness to hypoxia in sham rabbits but not in CHF rabbits. Conversely, L-158,809 decreases CB chemoreceptor responses to hypoxia in CHF rabbits but not in sham rabbits. In addition, mRNA and protein expression of the AT1 receptor and the tissue level of Ang II in the CB are significantly elevated in CHF rabbits. These results indicate that elevated Ang II–AT1 receptor signaling in the CB in CHF contributes to an enhanced CB chemoreceptor sensitivity to hypoxia and thus augments peripheral chemoreflex function in CHF.
Previous studies have shown that chemoreflex sensitivity is increased in patients with CHF [14]. Studies from our laboratory have also found that in the rabbit model of pacing-induced CHF, peripheral chemoreflex sensitivity is enhanced, compared with that in sham rabbits [9]. This enhanced sensitivity of the peripheral chemoreflex contributes, at least in part, to the sympathetic activation in this rabbit model of CHF, because inhibition of peripheral chemoreceptor activity by inhalation of 100% O2 decreased RSNA in CHF, but not in sham rabbits [9]. However, the mechanisms involved in enhancing peripheral chemoreceptor sensitivity during CHF have not been clearly defined.
A variety of humoral substances have been shown to be elevated in the CHF state [15–17]. Ang II has been considered as a prime candidate that modulates sympathetic outflow in the CHF state, because it is known that Ang II alters sympathetic function at the central nervous system [18]. Previous studies have documented a role of central Ang II in enhancing sympathetic outflow in CHF [19]. The present study documents for the first time, that elevated circulating Ang II in conscious rabbits (at levels equivalent to the endogenous plasma Ang II level in CHF rabbits) enhances hypoxia-induced chemoreflex activation of RSNA. This effect of Ang II to enhance peripheral chemoreflex function in normal animals is similar to the enhanced chemoreflex function observed in CHF animals in which Ang II levels are similarly elevated. Furthermore, blockade of AT1 receptors by L-158,809 in CHF rabbits attenuated or normalized the exaggerated hypoxia-induced chemoreflex responses. Thus, the ability of Ang II to enhance peripheral chemoreflex function is likely to contribute, along with its other known sympathetic excitatory effects, to the sympathetic activation observed in CHF.
The loci at which Ang II acts within the reflex arc to enhance peripheral chemoreflex function remain to be fully examined. However, our results confirm previous studies showing that a functional AT1 receptor in the CB enhances chemoreceptor afferent activity [6,20]. In addition, angiotensinogen and Ang converting enzyme, but not renin, are expressed in CB tissue [7]. These results suggest that locally produced Ang II in the CB is capable of altering CB chemoreceptor function. Indeed, we found that the Ang II concentration and mRNA and protein expressions of AT1 receptor was increased in CBs from CHF rabbits. At the same time, the sensitivity of CB chemoreceptors to hypoxia was enhanced in CHF rabbits. It is significant that this enhanced chemoreceptor activity in CHF animals was observed from the isolated–perfused carotid sinus preparation devoid of circulating Ang II. We further found that blockade of AT1 receptors in the CB (without exposure to circulating Ang II) decreased CB chemoreceptor responses to hypoxia in CHF rabbits but not in sham rabbits (Fig. 5). These data clearly suggest that elevation of local tissue Ang II with the upregulation of AT1 receptors in the CB plays a major role in enhancing CB chemoreceptor sensitivity to hypoxia in CHF rabbits. Further studies are needed to determine whether other components of the local Ang II system are upregulated in the CB in CHF.
It appears, however, that Ang II cannot explain all of the changes in CB chemoreceptor activity that occur in CHF. The present study confirms our previous observation that CB chemoreceptor activity under normoxic (baseline) conditions is elevated in CHF rabbits. However, the present study demonstrates that perfusion of the CB with neither Ang II nor L-158,809 affects baseline CB chemoreceptor activity in either sham or CHF rabbits. On the other hand, Allen et al. have shown that perfusion of the CB with Ang II induces excitation of rat CB chemoreceptor activity under normoxic conditions [6]. The heterogeneity of these results could be due to the difference in the concentration Ang II employed. In Allen's study, a dose-dependent excitation of CB chemoreceptor activity was induced by Ang II with a threshold concentration of 1nM [6], but in our present study, 100pM Ang II (close to the endogenous plasma Ang II level in CHF rabbits) was used. Although it is possible that very high levels of Ang II may contribute to an elevated basal CB chemoreceptor activity in the normoxic state in CHF rabbits, it is likely that other mechanisms are responsible for this effect, such as a decrease in CB nitric oxide production, as we have shown previously [10].
The mechanism by which Ang II enhances the CB chemoreceptor sensitivity to hypoxia is not known. One possible mechanism is a reactive oxygen species (ROS) signaling pathway. Indeed, Ang II recently has been implicated in activation of ROS, specifically superoxide anion, in CHF [21]. Ang II stimulated superoxide anion production is linked to AT1 receptor activation, which then promotes activation of NADPH oxidase [22]. An interaction of Ang II and NADPH oxidase on neural control of cardiovascular function in CHF has been implicated [11]. Our preliminary work has found that the mRNA and protein expression of NADPH oxidase and the production of superoxide are enhanced in CBs from CHF rabbits and influence chemoreceptor sensitivity [23]. These results suggest that the NADPH–superoxide pathway may mediate the excitatory effect of Ang II in the CB.
Concerning the vasoconstrictor effect of Ang II, which is known to be influenced by an NADPH oxidase mechanism [24], the question arises whether Ang II, at least in part, enhances CB chemoreceptor sensitivity by reducing blood flow to the CB. We cannot discount the possibility that CB flow may have been altered by Ang II in the present study despite that fact that the isolated carotid sinus region was perfused at constant flow. Nevertheless, we (Fig. 3) and others [20] have shown that AT1 receptors are expressed on CB glomus cells, the initial site of sensory transduction in the CB [25]. In addition, in other preliminary studies [26], we have found that Ang II enhances the sensitivity of K+ currents to hypoxia in isolated glomus cells. Thus there is evidence to support an effect of Ang II on chemoreceptor function independent of the vasculature.
The present study demonstrates that the sympathoexcitatory effects of Ang II extend beyond that of central influences. Its ability to enhance afferent input from carotid body chemoreceptors provides another avenue in which to alter the balance of reflex control of sympathetic outflow toward that of higher sympathetic tone. In disease states such as heart failure, Ang II is likely to be playing a role at all levels of sympathetic function, including afferent as well as central and efferent mechanisms.
In conclusion, the results in this study indicate that the elevation of endogenous Ang II in CHF rabbits contributes to the enhanced peripheral chemoreflex control of sympathetic outflow seen in this disease state. Furthermore, our study demonstrates that Ang II in CHF acts at the level of the CB to enhance CB chemoreceptor sensitivity to hypoxia.
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Acknowledgements |
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The authors wish to thank Liu Xuefei, Kaye Talbitzer, and Anding Phyllis for their technical assistance, and Dr. Kurtis Cornish for his surgical assistance. This study was supported by a Program Project Grant from the Heart, Lung and Blood Institute of NIH (PO1-HL62222).
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Notes |
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Time for primary review 26 days
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