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Circulation Research. 2005;97:260-267
Published online before print June 30, 2005, doi: 10.1161/01.RES.0000175722.21555.55
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(Circulation Research. 2005;97:260.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Gene Transfer of Neuronal Nitric Oxide Synthase to Carotid Body Reverses Enhanced Chemoreceptor Function in Heart Failure Rabbits

Yu-Long Li, Yi-Fan Li, Dongmei Liu, Kurtis G. Cornish, Kaushik P. Patel, Irving H. Zucker, Keith M. Channon, Harold D. Schultz

From the Department of Cellular and Integrative Physiology (Y.-L.L., D.L., K.G.C., K.P.P., I.H.Z., H.D.S.), University of Nebraska Medical Center, Omaha; Division of Basic Biomedical Sciences (Y.-F.L.), University of South Dakota School of Medicine, Vermillion; and the Department of Cardiovascular Medicine (K.M.C.), University of Oxford, John Radcliffe Hospital, UK.

Correspondence to Harold D. Schultz, Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850. E-mail hschultz{at}unmc.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Our previous studies showed that decreased nitric oxide (NO) production enhanced carotid body (CB) chemoreceptor activity in chronic heart failure (CHF) rabbits. In the present study, we investigated the effects of neuronal NO synthase (nNOS) gene transfer on CB chemoreceptor activity in CHF rabbits. The nNOS protein expression and NO production were suppressed in CBs (P<0.05) of CHF rabbits, but were increased 3 days after application of an adenovirus expressing nNOS (Ad.nNOS) to the CB. As a control, nNOS and NO levels in CHF CBs were not affected by Ad.EGFP. Baseline single-fiber discharge during normoxia and the response to hypoxia were enhanced (P<0.05) from CB chemoreceptors in CHF versus sham rabbits. Ad.nNOS decreased the baseline discharge (4.5±0.3 versus 7.3±0.4 imp/s at 105±1.9 mm Hg) and the response to hypoxia (18.3±1.2 imp/s versus 35.6±1.1 at 40±2.1 mm Hg) from CB chemoreceptors in CHF rabbits (Ad.nNOS CB versus contralateral noninfected CB respectively, P<0.05). A specific nNOS inhibitor, S-Methyl-L-thiocitrulline (SMTC), fully inhibited the effect of Ad.nNOS on the enhanced CB activity in CHF rabbits. In addition, nNOS gene transfer to the CBs also significantly blunted the baseline renal sympathetic nerve activity (RSNA) and the response of RSNA to hypoxia in CHF rabbits (P<0.05). These results indicate that decreased endogenous nNOS activity in the CB plays an important role in the enhanced activity of the CB chemoreceptors and peripheral chemoreflex function in CHF rabbits.


Key Words: nitric oxide • gene transfer • chemoreceptor • hypoxia • heart failure


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The endogenous production of nitric oxide (NO) plays an important role in cardiovascular homeostasis through its action on the central and peripheral autonomic nervous systems.1 Although NO plays a significant excitatory role in the nucleus tractus solitarii (NTS),2–4 many studies have shown that NO produced within the carotid body (CB) is an inhibitory modulator of CB chemoreceptor activity.5–11 For example, the administration of the precursor L-arginine, NO donor molecules,5,6,9 or NO gas8 to the cat CB perfused in vitro reduces the chemoreceptor response to hypoxia. Conversely, inhibition of nitric oxide synthase (NOS) increases the frequency of CB chemoreceptor discharge in situ and in vitro.5,12,13

Profound activation of the sympathetic nervous system is characteristic of chronic heart failure (CHF).14–16 Peripheral chemoreceptor activation is an excitatory input that increases sympathetic outflow.17 Peripheral chemoreceptor sensitivity is enhanced in both clinical and experimental CHF18–20 and contributes to the tonic elevation in sympathetic function. Our recent studies have shown that a decreased NO production is involved in the enhanced CB chemoreceptor activity in CHF.13 We found that NOS inhibition enhanced the sensitivity of the CB chemoreceptors13 and decreased the CB glomus cell outward potassium currents (IK)21 in sham rabbits but was without effect in CHF rabbits. Whereas, the NO donor (S-nitroso-N-acetyl-penicillamine [SNAP]) normalized the enhanced sensitivity of CB chemoreceptors and augmented glomus cell IK in CHF rabbits.13,21

At least 3 isoforms of NOS have been isolated :22 neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Histochemical studies in cat CB11 have demonstrated that intrinsic neurons innervating the intraglomic arterioles and glomus cells in addition to intraglomal vascular endothelial cells are positive for NOS (nNOS and eNOS). However, the contribution of nNOS and eNOS isoforms to the production of NO in the CB has not yet been adequately explored. Two studies concluded that a nonspecific NOS inhibitor, L-NAME, significantly enhanced the ventilatory response to NaCN in rat3 and the CB chemoreceptor response to hypoxia in cats;23 whereas specific nNOS inhibitors were ineffective.3,23 Conversely, Kline et al,24,25 using mutant mice deficient in nNOS and eNOS isoforms, found that mice lacking nNOS showed greater ventilatory responses to hypoxia than wild-type controls; whereas responses to hypoxia were blunted in mutant mice lacking eNOS compared with the wild-type. Until now, even less is known about which isoform of NOS contributes to the enhanced peripheral chemoreceptor activity in CHF rabbits. Therefore, in the present study, we investigated the effect of Ad.nNOS gene transfer to the CB on the enhanced peripheral chemoreceptor activity in CHF rabbits.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Pacemaker Implant and Production of CHF
All experiments were performed on male New Zealand White rabbits weighing 2.5 to 3.5 Kg. Experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were performed in accordance with the National Institutes of Health 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-hour dark-light cycle and fed standard rabbit chow (Harlan Techlab) with water available ad libitum.

Rabbits underwent sterile thoracic instrumentation and then were paced to induce CHF, as previously described.19 Rabbits with >40% reductions in dD/dtmax and shortening fraction were considered in CHF (generally after 3 to 4 weeks). Sham-operated animals underwent a similar period of sonographic measurements with the pacemaker turned off. Any rabbit exhibiting abnormal arterial blood gases (PaO2<85 mm Hg; 45 mm Hg < PaCO2<30 mm Hg) was excluded from the study. See online supplement available at http://circres.ahajournals.org for details of instrumentation and cardiac function analysis.

Gene Transfer with Ad.EGFP or Ad.nNOS to the CB
The Ad.nNOS originally described by Channon et al26 was used in these experiments. This Ad.nNOS, containing a rat nNOS cDNA, expresses functional nNOS protein when perfused in carotid arteries of rabbits.27 Three days before the experiment, using sterile surgical technique, the left and right carotid sinus regions were exposed via a small incision. The sinus region was temporarily vascularly isolated (including the common carotid artery, internal carotid artery, and external carotid artery), and the tip of a PE-10 catheter was positioned at the level of the carotid body via the external maxillary artery. After these arteries were occluded with snares, 200 µL of Ad.EGFP (as control adenoviruses) or Ad.nNOS (1x108 pfu/mL, dissolved in 0.9% sodium chloride28) was slowly injected into the carotid body via the catheter and the snares around the vessels were removed. A similar sham surgery, without adenoviral injection, was performed on the contralateral sinus region as a control in the same animal. In reflex experiments (see below), application of either Ad.EGFP or Ad.nNOS was performed on both right and left CBs in the same animal. The incision was closed, and the rabbits were placed on an antibiotic regimen consisting of 5 mg/kg Baytril i.m. daily. Ad.EGFP or Ad.nNOS showed no signs of damage (cell fragments) to the CB as observed from light microscopic evaluation of histological sections.

Examination of Infection Efficiency of the Adenoviruses and Immunofluorescence for nNOS Detection in the CB
Carotid bodies were obtained from sham (unpaced) and from CHF rabbits. Each rabbit was perfused transcardially with 500 mL heparinized saline followed by 1500 mL of freshly prepared 4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4).

For Ad.EGFP measurement (3 CHF rabbits), both CBs in each rabbit were rapidly removed. The CB was blocked in the coronal plane and sectioned at 30 µm thickness in a cryostat. The sections were mounted onto chrome-alum–coated slides. The slides were dried. EGFP was directly measured under a Leica microscope at 510 nm with single excitation peak and at 490 nm of green light29 to evaluate the infection efficiency of the adenovirus.30

For nNOS immunofluorescence detection (5 sham and 5 CHF rabbits), both CBs in each rabbit were rapidly removed and postfixed in 4% paraformaldehyde in 0.1 mol/L PBS for 12 hours at 4°C, followed by soaking the CBs in 30% of sucrose for 12 hours at 4°C for cryostat protection. The CB was cut into 30 µm–thick sections. The CB sections were mounted on precoated glass slides for immunofluorescence for nNOS detection (see online supplement for details).

Western Blot Analysis for nNOS in the CB
Carotid bodies from sham and CHF rabbits were rapidly removed and immediately frozen in dry ice and stored at –80°C until analyzed. The protein was extracted with the lysing buffer (10 mmol/L PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS) plus protease inhibitor cocktail (Sigma, 100 µL/mL). After a centrifugation at 12 000g for 20 minutes at 4°C, the protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce Chemical). The protein sample was used for Western blot analysis31 for nNOS (see online supplement for details).

NO Measurement in the CB
Homogenates were prepared from CB samples. Total protein concentration was determined using a BCA protein assay kit (Pierce Chemical). NO was measured using a gas-phase chemiluminescent method32 (NOA 280i, Sievers). See online supplement for details.

Recording of Afferent Discharge of CB Chemoreceptors
Single unit action potentials were recorded from CB chemoreceptor fibers in the carotid sinus nerve as we have described previously (8 sham and 16 CHF rabbits).20 Both sinus regions (adenoviral infected CB versus control CB) were vascularly isolated and perfused with Krebs-Henseleit solution (in mM: 120 NaCl, 4.8 KCl, 2.0 CaCl2, 2.5 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.1 L-arginine, and 5.5 glucose). Perfusate was bubbled with O2/CO2/N2 gas mixture. CB nerve recordings were performed 3 days after exposure to adenovirus. See online supplement for details.

Peripheral Chemoreflex Control of Renal Sympathetic Nerve Activity and Ventilation
Renal sympathetic nerve recording electrodes were implanted as we have described previously.19 At that time, arterial/venous catheters were inserted into the right carotid artery and jugular vein, and either Ad.EGFP or Ad.nNOS was injected into both right and left CBs in CHF rabbits as described above. Experiments (6 sham and 18 CHF rabbits) were performed 3 days after surgical instrumentation/adenoviral application.

Changes in renal sympathetic nerve activity (RSNA) and minute ventilation (VE) in response to stimulation of peripheral chemoreceptors were measured in sham and CHF rabbits in the conscious resting state as described in our previous study.19 Peripheral chemoreceptors were stimulated preferentially by allowing the rabbits to breathe graded mixtures of hypoxic gas under isocapnic conditions. See online supplement for details.

Statistical Analysis
All data are expressed as mean±SEM. Statistical significance was determined by a 2-way ANOVA, followed by a Bonferroni procedure for post-hoc analysis for multiple comparisons. Statistical significance was accepted when P<0.05.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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Induction of CHF
Rapid left ventricular pacing induced CHF by the third or fourth week of pacing. LV dD/dtmax and LV shortening fraction were reduced after 3 or 4 weeks of pacing, compared with prepared baseline (P<0.05) (data available in online supplement). There was no significant change in the LV dD/dtmax and LV shortening fraction from baseline during 4 weeks in sham rabbits.

Confirmation of Adenovirus Gene Transfer
The expression of EGFP was used to confirm the efficacy of adenovirus infection. EGFP was visible in the CB from CHF rabbits (n=3) infected with Ad.EGFP (Figure 1B). However, no EGFP was observed in the contralateral CB (without Ad.EGFP injection) from these same rabbits (Figure 1D). There was no expression of EGFP in the heart and brain of these animals (data not shown). These results confirm that our method for selective gene transfer to the CB is feasible.



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  		Figure 1. Adenoviral mediated transfer of enhanced green fluorescent protein (EGFP) to the CB in a CHF rabbit. A, Bright field monochrome image of CB infected with Ad.EGFP. B, The same field viewed in A as shown for EGFP (green immunofluorescent image). C, Bright field monochrome image of the contralateral control (noninfected) CB from the same rabbit. D, The same field viewed in C as shown for EGFP (green immunofluorescent image). Note absence of EGFP in the noninfected CB. The arrows indicate glomus cell clusters.

Expression of nNOS and NO Production in the CBs from CHF Rabbits After the Transfer of Ad.nNOS
Using immunohistochemical analysis, we found that the expression of endogenous nNOS was localized in nerve fibers in the CB (Figure 2). In addition, the expression of nNOS was lower in the CB from CHF rabbits than that from sham rabbits (Figure 2). Three days after injection of Ad.nNOS (200 µL, 1x108 pfu/mL) to the CB of CHF rabbits, the expression of nNOS in the CB (Figure 3B) was significantly increased, compared with that in the noninfected CB from the same animals (Figure 3A). However, Ad.EGFP did not affect the expression of nNOS in the CBs of CHF rabbits (Figure 3C and 3D).



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  		Figure 2. Endogenous nNOS expression in noninfected CBs from sham (A through C) and CHF (D through F) rabbits. A, Green immunofluorescent image for neuronal filament. B, Red immunofluorescent image for nNOS. C, Merged A and B images (yellow color) illustrating overlap of neuronal filament and nNOS in the CB from a sham rabbit. D through F, Images as in A through C, but from a noninfected CB from a CHF rabbit. Note the marked reduction in nNOS staining in the CHF CB (E) compared with sham (B).



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  		Figure 3. nNOS expression in CBs from CHF rabbits infected with Ad.nNOS or Ad.EGFP. A and C, nNOS expression (red immunofluorescent image) in the control (noninfected) CBs of 2 CHF rabbits. B, nNOS expression in the Ad.nNOS-infected contralateral CB of rabbit A. D, nNOS expression in the Ad.EGFP-infected contralateral CB of rabbit C. The arrows indicate glomus cell clusters.

We also used Western blot analysis to measure the protein expression of nNOS in each group. CHF markedly decreased the protein expression of endogenous nNOS in the CBs, compared with that in sham rabbits (Figure 4A and 4B). Ad.nNOS infection significantly enhanced the intensity of the bands of nNOS in the CBs from the CHF rabbits compared with that in the noninfected CBs from the CHF animals (Figure 4A and 4B). However, Ad.EGFP did not affect the protein expression of nNOS in the CBs of CHF rabbits (Figure 4A and 4B). The levels of nNOS protein expression in the CB measured by immunoblot (Figure 4A and 4B) are consistent with the degree of immunohistochemical staining of nNOS observed for each group (Figure 2 and 3Up).



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  		Figure 4. nNOS protein and NO production in treated and untreated CBs. A, Representative gel of nNOS and ß-tubulin proteins in sham (unpaced), CHF, CHF+Ad.nNOS, and CHF+Ad.EGFP-treated CBs. A positive nNOS protein control (brain paraventricular nucleus PVN) and negative (absence of primary antibody) control are shown on the same gel. B, Relative nNOS protein expression in sham, CHF, CHF+Ad.nNOS, and CHF+Ad.EGFP treated CBs. n=6 in each group. C, NO concentration in sham, CHF, CHF+Ad.nNOS, and CHF+Ad.EGFP-treated CBs. n=4 in each group. Data are mean±SEM, *P<0.05 vs sham; #P<0.05 vs CHF rabbits.

The NO concentration in CBs from CHF rabbits was significantly less than that in sham rabbits (Figure 4C). Ad.nNOS gene transfer restored NO production in the CBs from CHF rabbits to normal levels. Ad.EGFP had no effect on NO production in the CBs of CHF rabbits (Figure 4C).

Effect of Ad.nNOS on CB Chemoreceptor Activity in CHF Rabbits
Previously, we showed that the baseline discharge of CB chemoreceptors during normoxia and the response to isocapnic hypoxia were enhanced in CHF rabbits compared with sham rabbits.20 In the present study, we observed similar results (Table). After Ad.nNOS infection of the CB of CHF rabbits, CB chemoreceptor activity during normoxia and hypoxia was significantly blunted (Table and Figure 5) as compared with that from the noninfected CB in the same animals. Ad.EGFP showed no effect on CB chemoreceptor activity (Table).


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  		Table 1. Effects of Adenoviral Gene Transfer of nNOS and EGFP on CB Chemoreceptor Activity in CHF Rabbits



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  		Figure 5. Representative recordings of action potentials from CB chemoreceptors in a CHF rabbit. Left, Control (noninfected) CB. Right, Ad.nNOS-infected contralateral CB. DF indicates discharge frequency; AP, action potential.

S-Methyl-L-thiocitrulline (SMTC, a specific nNOS inhibitor; Cayman Chemical Company) increased CB chemoreceptor activity during normoxia and hypoxia in CBs from sham rabbits and in CBs infected with Ad.nNOS from CHF rabbits (Figure 6). However, CB chemoreceptor activity during normoxia and hypoxia was not altered by SMTC in noninfected CBs from CHF rabbits or in CBs infected with Ad.EGFP.



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  		Figure 6. Effect of SMTC (1 µmol/L) on the activity of CB chemoreceptors in sham, CHF, CHF+Ad.nNOS, and CHF+Ad.EGFP-treated CBs. Hypoxia: PaO2=40±2.4 mm Hg. Data are mean±SEM, n=8 in each group. *P<0.05 vs normoxia; #P<0.05 vs control. {dagger}P<0.05 vs CHF Control or CHF+Ad.EGFP control.

Effect of Ad.nNOS on RSNA, VE, and MBP in CHF rabbits
We observed that RSNA at rest (normoxia) and the RSNA responses to hypoxia were elevated in CHF rabbits compared with that in sham rabbits (Figure 7A and 7B), which is consistent with that in our previous study.19 The ventilatory response to hypoxia was also enhanced in CHF rabbits (see online Table II). Ad.nNOS infection of both CBs in CHF rabbits markedly reduced resting RSNA and the RSNA and VE responses to hypoxia. However, these reflex responses to hypoxia were not reduced to the level seen in sham rabbits (Figure 7A, online Table II). Bilateral CB Ad.EGFP infection did not alter the enhanced RSNA at normoxic and hypoxic states in CHF rabbits (Figure 7A).



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  		Figure 7. Effect of bilateral CB gene transfer with either Ad.nNOS or Ad.EGFP (2x107 pfu) on RSNA (A) and MBP (B) under normoxic and hypoxic states in CHF rabbits, as compared with RSNA and MBP responses in unpaced sham rabbits without gene transfer. Data are mean±SEM, n=6 for each group. *P<0.05 vs sham; #P<0.05 vs CHF.

MBP was lower (Figure 7B) and HR higher (online supplement) in CHF compared with sham rabbits, and neither was influenced by Ad.nNOS or Ad.EGFP treatment.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study showed that (1) the expression of nNOS and NO production was suppressed in the CB from CHF rabbits along with enhanced CB chemoreceptor activity; (2) Ad.nNOS gene transfer enhanced the expression of nNOS and NO production in the CB from CHF rabbits and reversed the enhanced CB chemoreceptor activity in CHF rabbits; (3) the specific nNOS inhibitor, SMTC, abolished the effect of Ad.nNOS on CB chemoreceptor activity; (4) localized Ad.nNOS gene transfer to the CBs lowered resting RSNA and reduced peripheral chemoreflex sensitivity in conscious CHF rabbits.

Adenovirus-mediated gene transfers have been reported previously.27,28,33 However, it can be difficult to transfect localized tissues and not to affect other tissues in in vivo experiments. Using Ad.EGFP to evaluate the adenovirus-mediated gene transfer, we found that gene transfer to the CB only induced the expression of Ad.EGFP in the CB region but not in the contralateral uninfected CB (Figure 1) and other tissues (heart and brain) from the same rabbits. The successful gene transfer to the CBs established the solid methodological foundation for investigating the role of nNOS expression in the CBs on peripheral chemoreflex function in CHF rabbits. The enhanced expression of nNOS in the CBs of CHF rabbits by gene transfer was confirmed by immunohistochemistry (Figure 3) and Western blot analysis (Figure 4). The dose and time period of the gene transfer technique we used in the present study was based on previous studies27,28 in which adenovirus-mediated gene expression was maximal without tissue injury when the dose of 2x107 pfu and the time course of 3 days were used.

The CBs are a pair of small arterial chemoreceptor organs, which sense blood PaO2, PaCO2, and pH, and reflexly influence cardiopulmonary function via primary afferent fibers of the carotid sinus nerve (CSN).34,35 Because rabbits lack functional aortic chemoreceptors,36,37 the peripheral chemoreflex is attributable mainly to the CBs in this species. Our previous studies have shown that CB chemoreflex sensitivity is enhanced in rabbits with CHF.19 This enhanced sensitivity of the CB chemoreflex contributes to the sympathetic activation in the CHF state because inhibition of CB chemoreceptor activity decreased resting RSNA in CHF but not in sham rabbits.19 Our studies have also demonstrated that a decrease in NO production in the CBs is involved in the enhanced CB chemoreceptor activity and peripheral chemoreflex function in CHF rabbits.19–21

The glomus cells in the CB are thought to be primary chemosensory transducers by releasing excitatory neurotransmitters that depolarize carotid sinus nerve afferents.38 We have previously demonstrated that IK is blunted in CB glomus in CHF rabbits.21 This effect is mainly attributable to the suppression of KCa2+ channel activity caused by decreased availability of NO. The KCa2+ channel facilitation by NO in glomus cells is mediated by cGMP-dependent protein kinase G.21,39 NO also inhibits L-type Ca2+ channels in glomus cells of the rabbit CB via a cGMP independent process.40 The ability of nNOS gene transfer to reduce CB chemoreceptor activity in CHF rabbits in the present study is consistent with these effects of NO on ion channel function in CB glomus cells.

Histochemical and immunological studies have demonstrated NOS enzymes in the CBs of mammals.11,22,41,42 The nNOS isoform is present in the intrinsic neurons innervating the intraglomic arterioles and glomus cells. Intraglomal vascular endothelial cells contain eNOS. Several studies have shown that a nonspecific NOS inhibitor, L-NAME, significantly enhances the ventilatory response to NaCN in rats3 and CB response to hypoxia in cats;23 but specific nNOS inhibitors were ineffective on them.3,23 Conversely, using mutant mice deficient in nNOS and eNOS isoforms, Kline et al24,25 found that mice lacking nNOS showed greater ventilatory responses to hypoxia than wild-type controls; whereas responses to hypoxia were blunted in mutant mice lacking eNOS compared with the wild-type.

Our study confirms previous studies showing the presence of nNOS in nerve fibers in the CB.5,22 Furthermore, our results demonstrate that nNOS protein expression and NO production are markedly lower in the CB from CHF rabbits compared with that in sham rabbits. Gene transfer of nNOS to the CB enhanced protein expression and NO production in the CB and reversed the enhanced CB chemoreceptor activity of CHF rabbits. The specific nNOS inhibitor, SMTC, abolished the effect of Ad.nNOS on CB chemoreceptor activity. Equally important, SMTC alone enhanced CB chemoreceptor activity in sham rabbits, indicating that, in this species, nNOS provides a tonic inhibitory influence on CB chemoreceptor activity under normal conditions. By contrast, SMTC failed to increase CB chemoreceptor activity in CHF rabbits without nNOS gene transfer, indicating a loss of this tonic inhibitory influence in the CHF state. These results, taken together, demonstrate that a marked down regulation of endogenous nNOS in the CB is involved in the enhanced CB chemoreceptor activity in CHF rabbits.

The adenoviral transfer of nNOS gene to the CB proved efficacious in elevating nNOS protein expression and NO production in treated CBs of CHF rabbits to levels found in sham rabbits. Yet, even though ad.nNOS treatment reduced CB chemoreceptor activity and chemoreflex function in CHF rabbits toward that seen in sham rabbits, the gene transfer did not completely normalize CB function. It is possible that the inability of this technique to target specific cell types within the CB influenced the efficacy of the gene transfer on chemoreceptor function. In addition, the role of endogenous eNOS on the CB chemoreceptor activity cannot be assessed from the present study.

Alternatively, other endogenous active substances besides NO (such as angiotensin II, Ang II) may also play a role in this pathophysiological process. In recent studies, we have found that Ang II enhanced the hypoxia-induced RSNA response in sham rabbits but not in CHF rabbits. Conversely, the AT1 receptor antagonist, L-158 809 attenuated hypoxia-induced increases in RSNA in CHF rabbits but not in sham rabbits.43 We also found that NADPH oxidase–derived superoxide anion mediated the Ang II-enhanced CB chemoreceptor activity in CHF rabbits.44 But the relationship among NO, Ang II, and superoxide anion on CB chemoreceptor function is not yet clear. Ang II may depress NOS gene expression45 and affect the bioavailability of NO via increasing endogenous superoxide anion production.46 Further study is needed to explore the mechanism of the enhanced CB chemoreceptor function in CHF rabbits that appears to be independent of, or interacts with, nNOS-derived NO.

In the present study, nNOS gene transfer to both CBs significantly blunted the enhanced RSNA at rest (normoxia) and during hypoxia in conscious CHF rabbits. These results demonstrate the important contribution of enhanced CB chemoreceptor input to elevated sympathetic outflow in CHF and the contribution of nNOS downregulation in the CB to this effect. The fact that enhanced gene expression of CB nNOS did not completely normalize peripheral chemoreflex function in CHF rabbits (Figure 7A) is expected given our observation that enhanced nNOS expression in the CB also did not completely normalize CB chemoreceptor activity in CHF rabbits (Table). In addition, it is well known that a number of other cardiovascular reflex and central neural alterations contribute to elevated sympathetic activity in CHF.47 Our results underscore the significance of a multiplicity of factors contributing to sympathetic hyperactivity in CHF.

In conclusion, we have described a model of nNOS gene transfer to the CBs for evaluating CB function. The present results demonstrate that the nNOS downregulation in the CB contributes to the enhanced CB chemoreceptor activity and the sympatho-excitation in the CHF state.


*    Acknowledgments
 
This study was supported by a Program Project Grant from the National Heart, Lung, and Blood Institute (PO-1 HL062222). The authors thank Denise Arrick and Kaye Talbitzer for their technical assistance and Dr Todd Wyatt for performing the NO measurements.


*    Footnotes
 
Original received January 26, 2005; revision received June 9, 2005; accepted June 21, 2005.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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