Gene Transfer of Neuronal Nitric Oxide Synthase to Carotid Body Reverses Enhanced Chemoreceptor Function in Heart Failure Rabbits
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循环研究杂志 2005年第8期
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
the Department of Cardiovascular Medicine (K.M.C.), University of Oxford, John Radcliffe Hospital, UK.
Abstract
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
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),2eC4 many studies have shown that NO produced within the carotid body (CB) is an inhibitory modulator of CB chemoreceptor activity.5eC11 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).14eC16 Peripheral chemoreceptor activation is an excitatory input that increases sympathetic outflow.17 Peripheral chemoreceptor sensitivity is enhanced in both clinical and experimental CHF18eC20 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
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 e蘈 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 e thickness in a cryostat. The sections were mounted onto chrome-alumeCcoated 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 eeCthick 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 eC80°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 e蘈/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
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.
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 e蘈, 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).
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 3).
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).
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.
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).
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
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.19eC21
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 oxidaseeCderived 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.
References
Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci U S A. 1991; 88: 7797eC7801.
Gozal D, Gozal E, Gozal YM, Torres JE. Nitric oxide synthase isoforms and peripheral chemoreceptor stimulation in conscious rats. Neuroreport. 1996; 7: 1145eC1148.
Haxhiu MA, Chang CH, Dreshaj IA, Erokwu B, Prabhakar NR, Cherniack NS. Nitric oxide and ventilatory response to hypoxia. Respir Physiol. 1995; 101: 257eC266.
Ruggiero DA, Mtui EP, Otake K, Anwar M. Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J Comp Neurol. 1996; 364: 51eC67.
Wang ZZ, Stensaas LJ, Bredt DS, Dinger B, Fidone SJ. Localization and actions of nitric oxide in the cat carotid body. Neuroscience. 1994; 60: 275eC286.
Alcayaga J, Iturriaga R, Ramirez J, Readi R, Quezada C, Salinas S. Cat carotid body chemosensory response to non-hypoxic stimuli is inhibited by sodium nitroprusside both in situ and in vitro. Brain Res. 1997; 767: 384eC387.
Chugh DK, Katayama M, Mokashi A, Debout DE, Ray DK, Lahiri S. Nitric oxide-related inhibition of carotid chemosensory nerve activity in the cat. Respir Physiol. 1994; 97: 147eC156.
Iturriaga R, Mosqueira M, Villanueva S. Effects of nitric oxide gas on carotid body chemosensory response to hypoxia. Brain Res. 2000; 855: 282eC286.
Iturriaga R, Villanueva S, Mosqueira M. Dual effects of nitric oxide on cat carotid body chemoreception. J Appl Physiol. 2000; 89: 1005eC1012.
Katayama M, Chugh DK, Mokashi A, Ray DK, Bebout DE, Lahiri S. NO mimics O2 in the carotid body chemoreception. Adv Exp Med Biol. 1994; 360: 225eC227.
Prabhakar NR, Kumar GK, Chang CH, Agani FH, Haxhiu MA. Nitric oxide in the sensory function of the carotid body. Brain Res. 1993; 625: 16eC22.
Iturriaga R, Alcayaga J, Rey S. Sodium nitroprusside blocks the cat carotid chemosensory inhibition induced by dopamine, but not that by hyperoxia. Brain Res. 1998; 799: 26eC34.
Sun SY, Wang W, Zucker IH, Schultz HD. Enhanced activity of carotid body chemoreceptors in rabbits with heart failure: role of nitric oxide. J Appl Physiol. 1999; 86: 1273eC1282.
Floras JS. Clinical aspects of sympathetic activation and parasympathetic withdrawal in heart failure. J Am Coll Cardiol. 1993; 22: 72AeC84A.
Esler M, Kaye D, Lambert G, Esler D, Jennings G. Adrenergic nervous system in heart failure. Am J Cardiol. 1997; 80: 7LeC14L.
Zucker IH, Wang W, Brandle M, Schultz HD, Patel KP. Neural regulation of sympathetic nerve activity in heart failure. Prog Cardiovasc Dis. 1995; 37: 397eC414.
Marshall JM. Peripheral chemoreceptors and cardiovascular regulation. Physiol Rev. 1994; 74: 543eC594.
Chua TP, Claek AL, Amadi AA, Coats AJ. Relation between chemosensitivity and the ventilatory response to exercise in chronic heart failure. J Am Coll Cardiol. 1996; 27: 650eC657.
Chugh SS, Chua TP, Coats AJ. Peripheral chemoreflex in chronic heart failure: friend and foe. Am Heart J. 1996; 132: 900eC904.
Sun SY, Wang W, Zucker IH, Schultz HD. Enhanced peripheral chemoreflex function in conscious rabbits with pacing-induced heart failure. J Appl Physiol. 1999; 86: 1264eC1272.
Li YL, Sun SY, Overholt JL, Prabhakar NR, Rozanski GJ, Zucker IH, Schultz HD. Attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide. J Physiol. 2004; 555: 219eC229.
Wang ZZ, Bredt DS, Fidone SJ, Stensaas LJ. Neurons synthesizing nitric oxide innervate the mammalian carotid body. J Comp Neurol. 1993; 336: 419eC432.
Valdees V, Mosqueira M, Rey S, Rio RD, Iturriaga R. Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms. Am J Physiol Lung Cell Mol Physiol. 2003; 284: L57eCL68.
Kline D, Yang T, Huang P, Prabhakar NR. Altered response to hypoxia in mutant mice deficient in neuronal nitric oxide synthase. J Physiol. 1998; 511: 273eC287.
Kline D, Yang T, Huang P, Premkumar DR, Thomas AJ, Prabhakar NR. Blunted respiratory responses to hypoxia in mutant mice deficient in nitric oxide synthase-3. J Appl Physiol. 2000; 88: 1496eC1508.
Channon KM, Blazing MA, Shetty GA, Potts KE, George SE. Adenoviral gene transfer of nitric oxidase synthase: high level expression in human vascular cells. Cardiovasc Res. 1996; 32: 962eC997.
Channon KM, Qian H, Neplioueva V, Blazing MA, Olmez E, Shetty GA, Youngblood SA, Pawloski J, McMahon T, Stamler JS, George SE. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-Fed rabbits. Circulation. 1998; 98: 1905eC1911.
Li YF, Roy SK, Channon KM, Zucker IH, Patel KP. Effect of in vivo gene transfer of nNOS in the PVN on renal nerve discharge in rats. Am J Physiol Heart Circ Physiol. 2002; 282: H594eCH601.
Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey JK, Dec GW, Semigran MJ, Rosenzweig A. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci U S A. 1998; 95: 5251eC5256.
Xia H, Qi H, Li Y, Pei J, Barton J, Blackstad M, Xu T, Tao W. LATS1 tumor suppressor regulates G2/M transition and apoptosis. Oncogene. 2002; 21: 1233eC1241.
Roy SK, Kole AR. Transforming growth factor-beta receptor type II expression in the hamster ovary: cellular site(s), biochemical properties, and hormonal regulation. Endocrinology. 1995; 136: 4610eC4620.
Wyatt TA, Forget MA, Sisson JH. Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells. Am J Pathol. 2003; 163: 1157eC1166.
Wang Y, Patel KP, Cornish KG, Cannon KM, Zucker IH. nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure. Am J Physiol Heart Circ Physiol. 2003; 285: H1660eCH1667.
Eyzaguirre C, Fitzgerald RS, Lahiri S, Zapata P. Arterial chemoreceptors. In: Fishman AP, ed. Handbook of Physiology, The Cardiovascular System. Bethesda, MD: Am Physiol Soc; 1983; Section 3 (Vol II): 557eC621.
Fidone S, Gonzalez C. Initiation and control of chemoreceptor activity in the carotid body. In: Fishman AP, ed. Handbook of Physiology, The Respiratory System. Bethesda, MD: Am Physiol Soc; 1983; Section 3 (Vol II): 247eC312.
Chalmers JP, Korner PI, White SW. The relative roles of the aortic and carotid sinus nerves in the rabbit in the control of respiration and circulation during arterial hypoxia and hypercapnia. J Physiol. 1967; 188: 435eC450.
Verna A, Roumy M, Leitner LM. Loss of chemoreceptive properties of the rabbit carotid body after destruction of the glomus cells. Brain Res. 1975; 100: 13eC23.
Fidone S, Gonzalez C, Dinger B, Gomez-Nino A, Obeso A, Yoshizaki K. Cellular aspects of peripheral chemoreceptor function. In: Crystal RG, West JG, Barnes PJ, Cherniack S, Weibel ER, eds. The Lung. Scientific Formulations. New York: Raven Press; 1991: 1319eC1332.
Silva JM, Lewis DL. Nitric oxide enhances Ca(2+)-dependent K(+) channel activity in rat carotid body cells. Pflugers Arch. 2002; 443: 671eC675.
Summers BA, Overholt JL, Prabhakar NR. Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism. J Neurophysiol. 1999; 81: 1449eC1457.
Grimes PA, Mokashi A, Stone RA, Lahiri S. Nitric oxide synthase in autonomic innervation of the cat carotid body. J Auton Nerv Syst. 1995; 54: 80eC86.
Hohler B, Mayer B, Kummer W. Nitric oxide synthase in the rat carotid body and carotid sinus. Cell Tissue Res. 1994; 276: 559eC564.
Li YL, Xia XH, Li YF, Patel KP, Schultz HD. Upregulation of angiotensin II mediates the enhanced peripheral chemoreceptor sensitivity in heart failure rabbits. (Abstract) Circulation. Suppl. 2003; 108: IVeC241.
Li YL, Gao L, Wang W, Zucker IH, Schultz HD. NADPH oxidase-derived superoxide anion mediates the angiotensin II-enhanced peripheral chemoreceptor sensitivity in heart failure rabbits. (Abstract) Circulation. Suppl. 2004; 110: IIIeC265.
Kihara M, Umemura S, Kadota T, Yabana M, Tamura K, Nyuui N, Ogawa N, Murakami K, Fukamizu A, Ishii M. The neuronal isoform of constitutive nitric oxide synthase is up-regulated in the macula densa of angiotensinogen gene-knockout mice. Lab Invest. 1997; 76: 285eC294.
Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation. 1997; 95: 588eC593.
Zucker IH, Pliquett RU. Novel mechanisms of sympatho-excitation in chronic heart failure. Heart Fail Monit. 2002; 3: 2eC7.(Yu-Long Li, Yi-Fan Li, Do)
Division of Basic Biomedical Sciences (Y.-F.L.), University of South Dakota School of Medicine, Vermillion
the Department of Cardiovascular Medicine (K.M.C.), University of Oxford, John Radcliffe Hospital, UK.
Abstract
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
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),2eC4 many studies have shown that NO produced within the carotid body (CB) is an inhibitory modulator of CB chemoreceptor activity.5eC11 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).14eC16 Peripheral chemoreceptor activation is an excitatory input that increases sympathetic outflow.17 Peripheral chemoreceptor sensitivity is enhanced in both clinical and experimental CHF18eC20 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
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 e蘈 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 e thickness in a cryostat. The sections were mounted onto chrome-alumeCcoated 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 eeCthick 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 eC80°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 e蘈/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
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.
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 e蘈, 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).
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 3).
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).
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.
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).
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
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.19eC21
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 oxidaseeCderived 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.
References
Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci U S A. 1991; 88: 7797eC7801.
Gozal D, Gozal E, Gozal YM, Torres JE. Nitric oxide synthase isoforms and peripheral chemoreceptor stimulation in conscious rats. Neuroreport. 1996; 7: 1145eC1148.
Haxhiu MA, Chang CH, Dreshaj IA, Erokwu B, Prabhakar NR, Cherniack NS. Nitric oxide and ventilatory response to hypoxia. Respir Physiol. 1995; 101: 257eC266.
Ruggiero DA, Mtui EP, Otake K, Anwar M. Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J Comp Neurol. 1996; 364: 51eC67.
Wang ZZ, Stensaas LJ, Bredt DS, Dinger B, Fidone SJ. Localization and actions of nitric oxide in the cat carotid body. Neuroscience. 1994; 60: 275eC286.
Alcayaga J, Iturriaga R, Ramirez J, Readi R, Quezada C, Salinas S. Cat carotid body chemosensory response to non-hypoxic stimuli is inhibited by sodium nitroprusside both in situ and in vitro. Brain Res. 1997; 767: 384eC387.
Chugh DK, Katayama M, Mokashi A, Debout DE, Ray DK, Lahiri S. Nitric oxide-related inhibition of carotid chemosensory nerve activity in the cat. Respir Physiol. 1994; 97: 147eC156.
Iturriaga R, Mosqueira M, Villanueva S. Effects of nitric oxide gas on carotid body chemosensory response to hypoxia. Brain Res. 2000; 855: 282eC286.
Iturriaga R, Villanueva S, Mosqueira M. Dual effects of nitric oxide on cat carotid body chemoreception. J Appl Physiol. 2000; 89: 1005eC1012.
Katayama M, Chugh DK, Mokashi A, Ray DK, Bebout DE, Lahiri S. NO mimics O2 in the carotid body chemoreception. Adv Exp Med Biol. 1994; 360: 225eC227.
Prabhakar NR, Kumar GK, Chang CH, Agani FH, Haxhiu MA. Nitric oxide in the sensory function of the carotid body. Brain Res. 1993; 625: 16eC22.
Iturriaga R, Alcayaga J, Rey S. Sodium nitroprusside blocks the cat carotid chemosensory inhibition induced by dopamine, but not that by hyperoxia. Brain Res. 1998; 799: 26eC34.
Sun SY, Wang W, Zucker IH, Schultz HD. Enhanced activity of carotid body chemoreceptors in rabbits with heart failure: role of nitric oxide. J Appl Physiol. 1999; 86: 1273eC1282.
Floras JS. Clinical aspects of sympathetic activation and parasympathetic withdrawal in heart failure. J Am Coll Cardiol. 1993; 22: 72AeC84A.
Esler M, Kaye D, Lambert G, Esler D, Jennings G. Adrenergic nervous system in heart failure. Am J Cardiol. 1997; 80: 7LeC14L.
Zucker IH, Wang W, Brandle M, Schultz HD, Patel KP. Neural regulation of sympathetic nerve activity in heart failure. Prog Cardiovasc Dis. 1995; 37: 397eC414.
Marshall JM. Peripheral chemoreceptors and cardiovascular regulation. Physiol Rev. 1994; 74: 543eC594.
Chua TP, Claek AL, Amadi AA, Coats AJ. Relation between chemosensitivity and the ventilatory response to exercise in chronic heart failure. J Am Coll Cardiol. 1996; 27: 650eC657.
Chugh SS, Chua TP, Coats AJ. Peripheral chemoreflex in chronic heart failure: friend and foe. Am Heart J. 1996; 132: 900eC904.
Sun SY, Wang W, Zucker IH, Schultz HD. Enhanced peripheral chemoreflex function in conscious rabbits with pacing-induced heart failure. J Appl Physiol. 1999; 86: 1264eC1272.
Li YL, Sun SY, Overholt JL, Prabhakar NR, Rozanski GJ, Zucker IH, Schultz HD. Attenuated outward potassium currents in carotid body glomus cells of heart failure rabbit: involvement of nitric oxide. J Physiol. 2004; 555: 219eC229.
Wang ZZ, Bredt DS, Fidone SJ, Stensaas LJ. Neurons synthesizing nitric oxide innervate the mammalian carotid body. J Comp Neurol. 1993; 336: 419eC432.
Valdees V, Mosqueira M, Rey S, Rio RD, Iturriaga R. Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms. Am J Physiol Lung Cell Mol Physiol. 2003; 284: L57eCL68.
Kline D, Yang T, Huang P, Prabhakar NR. Altered response to hypoxia in mutant mice deficient in neuronal nitric oxide synthase. J Physiol. 1998; 511: 273eC287.
Kline D, Yang T, Huang P, Premkumar DR, Thomas AJ, Prabhakar NR. Blunted respiratory responses to hypoxia in mutant mice deficient in nitric oxide synthase-3. J Appl Physiol. 2000; 88: 1496eC1508.
Channon KM, Blazing MA, Shetty GA, Potts KE, George SE. Adenoviral gene transfer of nitric oxidase synthase: high level expression in human vascular cells. Cardiovasc Res. 1996; 32: 962eC997.
Channon KM, Qian H, Neplioueva V, Blazing MA, Olmez E, Shetty GA, Youngblood SA, Pawloski J, McMahon T, Stamler JS, George SE. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-Fed rabbits. Circulation. 1998; 98: 1905eC1911.
Li YF, Roy SK, Channon KM, Zucker IH, Patel KP. Effect of in vivo gene transfer of nNOS in the PVN on renal nerve discharge in rats. Am J Physiol Heart Circ Physiol. 2002; 282: H594eCH601.
Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey JK, Dec GW, Semigran MJ, Rosenzweig A. Modulation of ventricular function through gene transfer in vivo. Proc Natl Acad Sci U S A. 1998; 95: 5251eC5256.
Xia H, Qi H, Li Y, Pei J, Barton J, Blackstad M, Xu T, Tao W. LATS1 tumor suppressor regulates G2/M transition and apoptosis. Oncogene. 2002; 21: 1233eC1241.
Roy SK, Kole AR. Transforming growth factor-beta receptor type II expression in the hamster ovary: cellular site(s), biochemical properties, and hormonal regulation. Endocrinology. 1995; 136: 4610eC4620.
Wyatt TA, Forget MA, Sisson JH. Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells. Am J Pathol. 2003; 163: 1157eC1166.
Wang Y, Patel KP, Cornish KG, Cannon KM, Zucker IH. nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure. Am J Physiol Heart Circ Physiol. 2003; 285: H1660eCH1667.
Eyzaguirre C, Fitzgerald RS, Lahiri S, Zapata P. Arterial chemoreceptors. In: Fishman AP, ed. Handbook of Physiology, The Cardiovascular System. Bethesda, MD: Am Physiol Soc; 1983; Section 3 (Vol II): 557eC621.
Fidone S, Gonzalez C. Initiation and control of chemoreceptor activity in the carotid body. In: Fishman AP, ed. Handbook of Physiology, The Respiratory System. Bethesda, MD: Am Physiol Soc; 1983; Section 3 (Vol II): 247eC312.
Chalmers JP, Korner PI, White SW. The relative roles of the aortic and carotid sinus nerves in the rabbit in the control of respiration and circulation during arterial hypoxia and hypercapnia. J Physiol. 1967; 188: 435eC450.
Verna A, Roumy M, Leitner LM. Loss of chemoreceptive properties of the rabbit carotid body after destruction of the glomus cells. Brain Res. 1975; 100: 13eC23.
Fidone S, Gonzalez C, Dinger B, Gomez-Nino A, Obeso A, Yoshizaki K. Cellular aspects of peripheral chemoreceptor function. In: Crystal RG, West JG, Barnes PJ, Cherniack S, Weibel ER, eds. The Lung. Scientific Formulations. New York: Raven Press; 1991: 1319eC1332.
Silva JM, Lewis DL. Nitric oxide enhances Ca(2+)-dependent K(+) channel activity in rat carotid body cells. Pflugers Arch. 2002; 443: 671eC675.
Summers BA, Overholt JL, Prabhakar NR. Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism. J Neurophysiol. 1999; 81: 1449eC1457.
Grimes PA, Mokashi A, Stone RA, Lahiri S. Nitric oxide synthase in autonomic innervation of the cat carotid body. J Auton Nerv Syst. 1995; 54: 80eC86.
Hohler B, Mayer B, Kummer W. Nitric oxide synthase in the rat carotid body and carotid sinus. Cell Tissue Res. 1994; 276: 559eC564.
Li YL, Xia XH, Li YF, Patel KP, Schultz HD. Upregulation of angiotensin II mediates the enhanced peripheral chemoreceptor sensitivity in heart failure rabbits. (Abstract) Circulation. Suppl. 2003; 108: IVeC241.
Li YL, Gao L, Wang W, Zucker IH, Schultz HD. NADPH oxidase-derived superoxide anion mediates the angiotensin II-enhanced peripheral chemoreceptor sensitivity in heart failure rabbits. (Abstract) Circulation. Suppl. 2004; 110: IIIeC265.
Kihara M, Umemura S, Kadota T, Yabana M, Tamura K, Nyuui N, Ogawa N, Murakami K, Fukamizu A, Ishii M. The neuronal isoform of constitutive nitric oxide synthase is up-regulated in the macula densa of angiotensinogen gene-knockout mice. Lab Invest. 1997; 76: 285eC294.
Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation. 1997; 95: 588eC593.
Zucker IH, Pliquett RU. Novel mechanisms of sympatho-excitation in chronic heart failure. Heart Fail Monit. 2002; 3: 2eC7.(Yu-Long Li, Yi-Fan Li, Do)