Published online before print May 21, 2007, doi: 10.1161/HYPERTENSIONAHA.107.088591
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(Hypertension. 2007;50:69.)
© 2007 American Heart Association, Inc.
Original Articles |
Noradrenergic Cell Specific Gene Transfer With Neuronal Nitric Oxide Synthase Reduces Cardiac Sympathetic Neurotransmission in Hypertensive Rats
From the Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy & Genetics, Oxford, UK.
Correspondence to David J. Paterson DPhil, DSc, Department of Physiology, Anatomy, and Genetics, Sherrington Building, Parks Road, Oxford, UK OX1 3PT. E-mail david.paterson{at}physiol.ox.ac.uk
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Abstract |
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Nitric oxide–cGMP pathway can inhibit cardiac norepinephrine (NE) release. Sympathetic hyper-responsiveness in hypertension may result from oxidative stress impairing this pathway. We tested the hypothesis that the gene transfer of neuronal NO synthase (nNOS) could restore sympathetic balance in the spontaneously hypertensive rat (SHR). An adenovirus (5x1010 particles) constructed with a noradrenergic neuron-specific promoter (PRS x8) encoding nNOS (Ad.PRS-nNOS) or enhanced green fluorescence protein (Ad.PRS-eGFP) was targeted to the right atrial wall by percutaneous injection in age-matched male SHRs and Wistar-Kyoto (WKY) rats. Five days after transduction, right atria were removed, and evoked [3H] norephinephrine (NE) release, NOS activity, and cGMP were measured. In the Ad.PRS-eGFP treated group, tissue levels of cGMP were significantly lower in the SHR compared with the WKY atria. NE release was also greater in the SHR, and soluble guanylate cyclase inhibition did not alter evoked [3H] NE release in the Ad.PRS-eGFP–treated SHR. All atria treated with Ad.PRS-nNOS had enhanced nNOS activity when compared with Ad.PRS-eGFP atria. Ad.PRS-nNOS in WKY rats reduced NE release compared with the Ad.PRS-eGFP group. Guanylate cyclase inhibition enhanced NE release in both Ad.PRS-nNOS– and Ad.PRS-eGFP–treated WKY atria. Ad.PRS-nNOS restored cGMP levels in the SHR to those seen in the WKY atria. In the SHR, Ad.PRS-nNOS also attenuated NE release compared with Ad.PRS-eGFP group. This was reversed by guanylate cyclase inhibition. We conclude that artificial upregulation of sympathetic nNOS via gene transfer with a noradrenergic promoter may provide a novel approach for correcting peripheral sympathetic hyperactivity in hypertension.
Key Words: nitric oxide • gene transfer • autonomic nervous system • norepinephrine • hypertension • cyclic GMP
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Introduction |
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In hypertension, cardiac autonomic impairment is an important contributor to the pathophysiological phenotype of the disease. Much evidence supports the observation that the activity of the sympathetic nervous system increases dramatically both in hypertensive patients1–4 and in the spontaneously hypertensive rat (SHR).5,6 The mechanism causing enhanced neurohumoral activation is unknown, but may be directly related to oxidative stress caused by hypertension disrupting nitric oxide (NO)–cGMP modulation of norepinephrine (NE) release in central autonomic nuclei7 and peripheral varicosities.8 Neuronal production of NO affects cardiac excitability through its action as a neuromodulator of postganglionic sympathovagal transmission.8–10 Both endogenous and exogenous NO inhibits evoked NE release during cardiac sympathetic nerve stimulation in the rat in vitro.11 Functionally, NO or the membrane-permeable cGMP analogue 8-bromo-cGMP decreases the heart rate response to sympathetic nerve stimulation in vitro, whereas NO synthase (NOS) inhibitors causes the opposite effect.12 In addition, oxidative stress caused by intermittent hypoxia decreases cardiac sympathetic neuronal NOS (nNOS) expression, which is associated with an enhanced heart rate response to sympathetic activation.13 Moreover, soluble guanylate cyclase (sGC), the key precursor of cGMP-dependent effects of NO, is downregulated in the aorta14 and the atria15 of the SHR. Tetrahydrobiopterin (BH4) and total biopterin levels are not different between SHR and Wistar-Kyoto (WKY) rat atria where sympathetic nerves innervate the heart, suggesting that BH4 is not uncoupled from NOS.15 Therefore strategies aimed at increasing bioavailability of NO could be advantageous in rescuing this impaired neural phenotype.
Gene transfer with adenoviral vectors is highly effective in modifying gene expression and has been used with good effect to understand whether changes in protein expression or enzyme activity play a key role in physiological function.16–18 Because adenoviruses transduce a broad range of cells, the gene of interest can be placed into cells that may not constitutively express them, thereby complicating the interpretation of the data. To minimize this limitation, we have recently developed a novel adenoviral vector constructed with a noradrenergic neuron-specific promoter (PRS x8) to drive nNOS or enhanced green fluorescence protein (eGFP) expression, and showed that nNOS targeted to only cardiac sympathetic neurons can inhibit noradrenergic neurotransmission and increase nNOS activity in normotensive rats.19
We therefore examined the hypothesis that a significant component of cardiac sympathetic hyper-responsiveness in hypertension occurs at the end organ level because of impaired NO-cyclic nucleotide signaling. From this we further hypothesized that gene transfer with a noradrenergic cell specific promoter coupled to nNOS could increase the bioactivity of nNOS and the production of cGMP that results in restoration of sympathetic transmission in the SHR to similar responses seen in the WKY rat.
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Materials and Methods |
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An extended methods and results section can be found in the online data supplement available at http://hyper.ahajournals.org.
Animals
Age-matched (16 to 20 weeks old) male SHR (n=28) and WKY rats (n=28), young Sprague-Dawley (SD) rats (8 to 13 days old; n=6) were purchased from Harlan (Bicester, UK) and housed under standard laboratory conditions. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the Animals (Scientific Procedures) Act 1986 (UK) and were performed under British Home Office license requirements (PPL 30/2130).
Gene Transfer Procedure
Replication-deficient adenoviral vectors encoding recombinant eGFP or nNOS under control of the noradrenergic neuron-specific promoter (PRS x8) were generated as described previously.19 Targeted percutaneous gene transfer to the right atrium was performed under isoflurane (Isocare, Animalcare Ltd) anesthesia (4% for induction and 2% to 3% for maintenance, in 100% O2), using a technique similar to that described previously for the guinea pig.9 SHRs or WKY rats received an injection of 5x1010 particles of Ad.PRS-nNOS or Ad.PRS-eGFP in 300 µL of phosphate-buffered saline. Experiments were performed 
5 days after injection.
[3H]Norepinephrine Release Experiments on the Isolated Right Atrium
An isolated right atrium preparation was generated by the method described previously20 with some modification. Details and experimental protocols may be found in the online data supplement.
Immunohistochemistry
Cardiac sympathetic neuron isolation and transduction was prepared by the method described previously.19 Detailed methods for immunohistochemistry may be found in the online data supplement.
Measurement of Tissue cGMP Levels and NOS Activity
Details may be found in the online data supplement.
Solutions and Drugs
Rat Tyrode solution contained (in mmol/L): NaCl 120, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2, and glucose 11. The solution was constantly aerated with carbogen to maintain pH at 7.4. All solutions were prepared fresh on the day of use using deionized water obtained from an Elga water purification system. Experiments using SNP were performed in a darkened room because of the light sensitivity of this drug.
Statistical Analysis
The results are given as the means±SEM. Analysis was performed using the paired or unpaired Student t test as appropriate. For all experiments, statistical significance was accepted at P<0.05.
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Results |
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Norepinephrine Release Was Increased in the SHR
Field stimulation-evoked release of [3H] NE from right atria was significantly enhanced at 5-Hz in the SHR (S1: +2.22±0.13%, S2: +2.19±0.14%, n=5) compared with the WKY rats (S1: +1.68±0.04%, S2: +1.55±0.04, n=6, P<0.01, unpaired t test; Figure 1). S1 and S2 represent the first and second field stimulation, respectively, where there was no difference between S1 and S2 in both strains indicating no significant time dependent changes.
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Effects of SNP and ODQ on Release of Norepinephrine in Field Stimulation
To determine whether the sympathetic activated NO–cGMP pathway was impaired in the SHR, the exogenous NO donor SNP and the sGC inhibitor ODQ were applied to preparations. SNP (20 µmol/L) significantly decreased the release of [3H] NE in response to 5-Hz field stimulation in the WKY (from 1.63±0.09% to 1.28±0.10%, n=6, P<0.01, paired t test, Figure 2), whereas [3H] NE release from SHR atria (n=6) was unaffected by SNP. Conversely, ODQ (10 µmol/L) enhanced the release of [3H] NE in the WKY (from 1.60±0.07% to 1.82±0.11%, n=5, P<0.01, paired t test; Figure 3), but did not affect release in the SHR (n=6).
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Sympathetic Neuron Specific Gene Transfer Mediated by Ad.PRS-nNOS
Majority of cultured sympathetic neurons did not have detectable amounts of endogenous nNOS. However, after 5 days after transduction with Ad.PRS-nNOS, nNOS was clearly detectable by nNOS antibody in 90% of sympathetic neurons (Figure 4A). nNOS expression in Ad.PRS-nNOS transduced cells colocalized with anti-TH stain (Figure 4B; scale bar, 50 µm).
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nNOS Activity After Ad.PRS-nNOS
We observed there was no difference in nNOS activity between SHR (n=9) and WKY (n=9) atria. After gene transfer with Ad.PRS-nNOS, nNOS activity was significantly enhanced in both strains compared with Ad.PRS-eGFP–treated atria (eg, SHR, increased from 14.6±2.3 to 20.5±3.1 fmol/mg/min, P<0.05; WKY, increased from 12.9±0.4 to 16.1±1.2 fmol/mg/min, P<0.05, n=6 in each group, Figure 5).
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Production of cGMP in Atria After Ad.PRS-nNOS
We observed cGMP concentration was significantly reduced in SHR compared with WKY tissue both in untreated tissue (3.57±0.11 versus 4.50±0.36 pmol/mg protein, P<0.05, unpaired t test, n=6 in each strain) and after gene transfer with Ad.PRS-eGFP (3.70±0.11 versus 4.37±0.17 pmol/mg protein, P<0.05, unpaired t test, n=6 in each strain; Figure 6). However, overexpression of Ad.PRS-nNOS reversed the attenuated production of cGMP in SHR (5.06±0.52 pmol/mg protein, n=6, P<0.05, unpaired t test) compared with untreated or Ad.PRS-eGFP–treated SHR.
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Effects of Gene Transfer With Ad.PRS-nNOS on Release of Norepinephrine
Previously, we showed that the Ad.PRS-nNOS gene transfer significantly reduced norephinephrine release evoked by field stimulation of isolated right atria in the normotensive SD rat.19 In the present study, we observed similar results in the WKY rats (Figure 7A). Ad.PRS-nNOS (1.49±0.05%, n=6) caused 16.1±3.1% reduction in the evoked NE release compared with Ad.PRS-eGFP–treated tissue (1.77±0.02%, n=5, P<0.01, unpaired t test). sGC inhibitor ODQ (10 µmol/L) also significantly enhanced the sympathetic responsiveness in the WKY rats treated with either the Ad.PRS-nNOS (1.67±0.06%) or Ad.PRS-eGFP–treated tissue (2.09±0.08%) (P<0.01 respectively, unpaired t test). In the SHR (Figure 7B), overexpression of Ad.PRS-nNOS (1.59±0.08%, n=6) significantly decreased the [3H] NE release by 21.2±4.0% compared with Ad.PRS-eGFP (2.02±0.17%, n=5, P<0.05, unpaired t test). There was no effect of ODQ on Ad.PRS-eGFP–treated SHR atria. However, ODQ significantly increased the NE release from Ad.PRS-nNOS–transfected SHR atria (1.89±0.11%, n=6, P<0.05, paired t test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we report 3 novel findings. First, a significant component of the sympathetic hyper-responsiveness in the SHR occurs postganglionically at the end organ level with enhanced evoked norepinephrine release. Secondly, this response was associated with impaired sGC-cGMP signaling in the SHR compared with the WKY rat. Thirdly, targeted gene transfer of nNOS with a noradrenergic cell specific promoter increased the bioactivity of nNOS and cGMP levels in cardiac sympathetic nerves. This resulted in decreased NE release in the SHR to levels seen in the normotensive control.
Peripheral Sympathetic Activity in the SHR
Autonomic imbalance in the central and peripheral nervous system has been implicated in the etiology of hypertension, and is also seen after myocardial infarction and heart failure.21–24
Impaired cardiac parasympathetic regulation and enhanced sympathetic activity characterize cardiac disease states, and are regarded as an independent predictor of mortality.25,26 Bradycardia and acetylcholine release in response to vagal nerve stimulation is reduced in the SHR at the level of the cardiac postganglionic neurons.27 Here we observed that the norepinephrine release from right atria in response to field stimulation in the SHR was significantly higher compared with the WKY rat. This gives direct evidence that the sympathetic nervous system is hyper-reactive in the SHR at the level of heart, and is in general agreement with whole animal studies that report a global sympathetic phenotype in this model of hypertension.28,29
NO–cGMP Signaling in the SHR
Defective NOS signaling via eNOS has been widely implicated in the vascular aspect of hypertension, although cumulative evidence now suggests that neuronally synthesized NO may also play a role in the pathophysiology of hypertension.30 The signaling pathway responsible for nNOS-derived NO inhibiting sympathetic neurotransmission is not completely understood. However, it could involve NO modifying cell physiology through activation of sGC and subsequent induction of cGMP production, which in turn activates cGMP-dependent protein kinase and phosphodiesterases that decrease cAMP-dependent phosphorylation of neuronal Ca2+ channels resulting in decreased exocytosis of neurotransmitter.31
Emerging data indicates that sGC, the main target protein for NO, is markedly desensitized/downregulated in hypertension.14,32–35 In particular, downregulation of the 
1 subunit of sGC in the atria and aorta of the SHR compared with WKY rat has recently been reported.15 We observed that guanylate cyclase inhibition increased NE release in the WKY but was without effect in the SHR, suggesting functional uncoupling of NO to its second messenger. Consistent with this idea we confirmed that tissue levels of cGMP were significantly lower in the SHR compared with the WKY. Translationally, decreased sGC expression and cGMP production has been proposed to account for impaired vasodilatation in the Afro-Caribbean population36 (susceptible to hypertension), fetal programming of hypertension,37 and in pulmonary hypertension.38 Similarly, downregulation of the sGC-dependent pathway34,35 has been demonstrated in aged and spontaneously hypertensive rats where superoxide production has been reported to trigger desensitization of vascular sGC in hypertension.34 Because nNOS activity and tetrahydrobiopterin levels in the SHR are not different compared with the WKY, this suggests uncoupling of nNOS from its main cofactor is unlikely to be the mechanism of peripheral sympathetic dysfunction in the SHR, and that the defect resides at the level of sGC-cGMP linkage.
Effect of Noradrenergic Neuron-Specific Overexpression of nNOS on Sympathetic Function in the SHR
Administration of the NO donor SNP did not decrease NE release in the SHR. It is possible that acute pharmacological administration of NO donors at a relatively low concentration (20 µmol/L) may not fully mimic the action of NO generated intracellularly by the nNOS transgene, given its labile and highly reactive properties. In particular, a relatively high local concentration of NO may be required to increase the bioavailability of NO under conditions of increased oxidative stress that have been demonstrated previously in hypertension.39,40 In addition, a much longer period of incubation may be need to achieve the effect than that can be provided in a stable in vitro experimental environment. However, overexpression of Ad.PRS-nNOS in the SHR significantly decreased the [3H] NE release compared with Ad.PRS-eGFP, and the sGC inhibitor ODQ significantly increased the NE release from Ad.PRS-nNOS–transduced SHR atria. The noradrenergic promoter is highly specific with no detectable leakage of gene expression into other cell types.19 Transduction with Ad.PRS-nNOS increased nNOS activity in the atrial extracts and enhanced atrial cGMP levels compared with Ad.PRS-eGFP. Importantly, gene transfer of adenovirus nNOS can also increase the expression of the defective 1 subunit of sGC in the SHR,27 which appears to be the key step for the functional changes. The mechanism underpinning this observation is not known, but when results are taken together they show that nNOS gene transfer provided a more potent NO signal than the NO donor.
Perspectives
Enhanced presynaptic postganglionic release of NE is not the only site of defective autonomic signaling in hypertension. nNOS gene transfer into the paraventricular nucleus can also decrease sympathetic outflow in the rat.41 In addition, our group15 recently found that the atria of the SHR were hyper-responsive to ß-adrenergic stimulation which was associated with elevated cAMP levels. This was accompanied by an increased basal and norepinephrine-stimulated L-type calcium current (ICaL) in single sinoatrial node cells. Gene transfer of nNOS decreased cAMP and the norepinephrine stimulated ICaL in the SHR. Combined with the present study, these results support the role of nNOS-sGC-cGMP in modulating peripheral noradrenergic activity at both presynaptic and postsynaptic levels, and that gene transfer of nNOS into either neurons or into pacemaking tissue can rescue the peripheral sympathetic hyperactivity seen in the SHR. Given the autocrine and paracrine action of NO in the heart, targeting both myocytes and sympathetic neurons with nNOS could be beneficial where this gaseous messenger may behave like a "smart beta blocker" that decreases overall adrenergic drive at these 2 sites. Whether this approach is viable to treat hyper-responsive sympathetic and beta adrenergic phenotypes remains to be established.
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Acknowledgments |
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We thank Chris Hirst for his technical assistance for setting up the experimental apparatus.
Sources of Funding
This work was supported by a grant from the British Heart Foundation (to D.L., D.J.P.), the Medical Research Council (to L.W., D.J.P.), and the Wellcome Trust (to C.-W.L., D.J.P.).
Disclosures
None.
Received February 5, 2007; first decision February 26, 2007; accepted May 2, 2007.
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