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(Hypertension. 2006;48:482.)
© 2006 American Heart Association, Inc.

Original Articles

Nox2, Ca²⁺, and Protein Kinase C Play a Role in Angiotensin II-Induced Free Radical Production in Nucleus Tractus Solitarius

Gang Wang; Josef Anrather; Michael J. Glass; M. Jacqueline Tarsitano; Ping Zhou; Kelly A. Frys; Virginia M. Pickel; Costantino Iadecola

From the Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY.

Correspondence to Gang Wang, Division of Neurobiology, Weill Medical College of Cornell University, 411 East 69th St, New York, NY 10021. E-mail gaw2001{at}med.cornell.edu

	Abstract

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The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca²⁺ currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II—induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca²⁺ or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC₅₀=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca²⁺ currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca²⁺ attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca²⁺ currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca²⁺ currents that underlie the actions of Ang II on central autonomic regulation.

Key Words: arterial hypertension • baroreflex • calcium channels • oxidative stress • blood pressure • autonomic nervous system

	Introduction

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A select group of brain stem nuclei regulates the systemic circulation by modulating cardiac output, vascular resistance, and fluid balance.^1,2 In particular, the dorsomedial region of the nucleus of the solitary tract (dmNTS), wherein vagal afferents from aortic baroreceptors and cardiorespiratory chemoreceptors terminate, plays a major role in cardiovascular regulation.^3–5 There is increasing evidence that angiotensin II (Ang II) is a critical neuromodulator in central autonomic nuclei,^6–9 including the dmNTS.^10–13 Within the dmNTS, activation of Ang II type 1 (AT₁) receptors alters cardiorespiratory reflexes including baroreceptor excitability and ion channel permeability.^10,14 These changes may contribute to Ang II-induced sympathoexcitation,^15–18 hypertension,^15,16 and heart failure.^17,18

Superoxide generated by the enzyme NADPH oxidase has emerged as a key intermediary in the central and peripheral effects of Ang II.^{10,14–16,18–21} NADPH oxidase, initially described in neutrophils,^22,23 is now known to be present in diverse cell types, including neurons.^{15,16,24–26} NADPH oxidase is composed of membrane-bound subunits, gp91^phox and p22^phox, and cytoplasmic subunits, p47^phox, p40^phox, p67^phox, and the small GTPase Rac1 and/or Rac2.^{22,23,27–30} The catalytic subunit gp91^phox, also termed Nox2, has several homologues, Nox1 and Nox3 through Nox5, the location of which is cell-type specific.^22,23 On stimulation of AT₁ receptors by Ang II, p47^phox is phosphorylated resulting in the assembly of the enzyme and production of superoxide.^23,31,32 In some cell types, protein kinase C (PKC) activation via intracellular Ca²⁺ is a critical step in p47^phox phosphorylation and subsequent enzyme assembly.^28,33,34

In dmNTS neurons, NADPH oxidase subunits are present in close association with AT₁ receptors.²⁵ Furthermore, reactive oxygen species (ROS) scavengers and NADPH oxidase inhibitors attenuate Ang II-elicited enhancement of Ca²⁺ currents in these neurons.²⁵ These findings have raised the possibility that NADPH oxidase-derived ROS are involved in the effects of Ang II on Ca²⁺ currents in dmNTS. However, direct evidence linking AT₁ receptors to NADPH oxidase-dependent ROS production through intracellular Ca²⁺ and PKC activation in dmNTS is missing. Furthermore, it is not known whether Nox2 is the catalytic subunit of NADPH oxidase that mediated the ROS production. In the present study, we used ROS imaging, whole-cell patch clamping, and Nox2-null mice to determine whether Ang II induces ROS production in dmNTS neurons and, if so, whether Nox2 plays a role in Ang II-induced ROS production and in the attendant changes in Ca²⁺ currents.

	Methods

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All of the experiments were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee at Weill Medical College of Cornell University.

Materials
Fluorescent dyes 4-(4-[didecylamino]styryl)- N-methylpyridinium iodide (DiA) and 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester [C-DCDHF-DA]) were purchased from Molecular Probes. Losartan was a gift from Merck and DuPont. Nox2-null mice were obtained from an in-house colony.^26,27 C57Bl/6J mice were used as wild-type (WT) controls. The polypeptide gp91ds-tat and its scrambled version³⁵ were synthesized by Bio-Synthesis.

Anterograde Labeling of NTS Neurons Receiving Vagal Afferents
Second-order vagal afferent neurons in the NTS were labeled with DiA in male Sprague-Dawley rats (2 to 4 weeks old) or in the male adult mice, as described previously.^25,36,37 Briefly, animals were anesthetized by a mixture of 8 mg/kg of ketamine and 8 mg/kg of xylazine. The right vagus nerve was isolated from the surrounding tissues, and a few crystals of DiA were placed on the nerve at the level of the carotid bifurcation and caudal to the nodose ganglion. The region was sealed with silicone elastomer (World Precision Instruments) to prevent dye leakage.

Dissociation of Second-Order NTS Neurons
Seven to 10 days after labeling, animals were euthanized by CO₂, and the brain stem was quickly removed and transferred to a chamber containing ice-cold sucrose-artificial cerebrospinal fluid.²⁵ Coronal slices were obtained and incubated with 0.02% pronase and thermolysin at 36°C in oxygenated lactic acid-containing artificial cerebrospinal fluid.²⁵ Using the area postrema as a landmark, the dmNTS was punched, and neurons were isolated. DiA-labeled presynaptic boutons from first-order neurons, remaining attached to second-order NTS neurons, were identified using a Nikon Diaphot 300 inverted fluorescence microscope. Unlabeled dmNTS neurons were also obtained from the contralateral NTS region of the same animal. The criteria for identification of the unlabeled dmNTS neurons were based on their anatomic location in the dorsomedial portion of the NTS and their typical cell morphology, for example, small round or oval bipolar cells with long thin processes.^25,36–40

Immunofluorescent Labeling
Dissociated dmNTS neurons were fixed in 4% paraformaldehyde. Neurons were permeabilized, treated with 3% BSA and incubated in a primary antisera mixture including rabbit anti-AT₁ receptor (1:100)^12,13,25 and goat anti-gp91^phox (1:50).²⁵ The neurons were then incubated in a mixture containing anti-rabbit Texas Red and anti-goat fluorescein isothiocyanate antisera (Jackson ImmunoResearch) and were visualized using the Nikon Diaphot 300 microscope.

Detection of Intracellular ROS
ROS production was assessed using C-DCDHF-DA.^41–43 C-DCDHF- DA loses its diacetate groups by cleavage via intracellular esterases and is oxidized by ROS to dichlorofluorescein (DCF). The isolated neurons were incubated with 5 µmol/L C-DCDHF-DA for 30 minutes. Time-resolved fluorescence was measured every 30 s using IPLab, an image analysis software from Sanalytics Inc. Recordings were started after a stable baseline was achieved. In all of the experiments, concurrent vehicle recordings were performed. No differences in the increase of DCF fluorescence induced by Ang II were observed between the DiA-labeled and DiA-unlabeled neurons selected from the contralateral dmNTS according to morphological criteria. Therefore, in all of the subsequent experiments, results from labeled and unlabeled neurons were pooled.

Electrophysiology
Voltage-gated Ca²⁺ currents were elicited using the whole-cell configuration of patch clamping.^25,39,40,44 An Axopatch-200A patch-clamp amplifier was used with the Cs⁺ electrode solution.²⁵ Using 2 mmol/L Ca²⁺ as a charge carrier, Ca²⁺ currents were elicited by 500-ms pulses from a holding potential of –60 mV to –20 mV using pClamp 8 (Axon Instruments).

Data Analysis
Data are expressed as mean±SEM. Paired or unpaired Student t test was performed. ROS data are expressed as the ratio Ft/Fo, where Ft is fluorescence after the application of Ang II in a given cell, and Fo is the baseline fluorescence of the same cell immediately before application of Ang II. Fo ranged from 105.2 to 169.7 relative fluorescence units. There was no relationship between baseline fluorescence (Fo) and the fluorescence increase induced by Ang II (Ft) under the experimental conditions studied.

	Results

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DiA-Labeled dmNTS Neurons Express AT₁ Receptors and Nox2
To determine whether AT₁ receptors and Nox2 were present in the same dmNTS neurons in which ROS were assessed, we examined AT₁ receptor and Nox2 immunoreactivity in DiA-labeled rat dmNTS neurons. DiA-labeled presynaptic boutons from first-order vagal afferent neurons were observed on isolated second-order dmNTS neurons (Figure 1A). The majority of DiA-labeled neurons examined were immunoreactive for AT₁ and/or Nox2 (Table). Thus, AT₁ receptors and Nox2 coexist in dmNTS neurons receiving vagal afferents.

View larger version (16K): [in this window] [in a new window]   Figure 1. Ang II potentiates ROS production in rat dmNTS neurons. A, AT1 receptors and Nox2 immunoreactivities are coexpressed in the same DiA-labeled dmNTS neuron. B, Ang II (100 nmol/L) increases DCF fluorescence in a DiA-labeled dmNTS neuron. C, Time course of DCF intensity in a single dmNTS neuron treated with vehicle (open symbol) or Ang II (closed symbol). D, Dose-response curve of the Ang II-induced potentiation of DCF (control: n=17; Ang II: 3 nmol/L, n=8; 10 nmol/L, n=8; 30 nmol/L, n=17; 100 nmol/L, n=17; 2000 nmol/L, n=16). Scale bars, 10 µm. *P<0.05 vs vehicle; **P<0.01 vs vehicle.

View this table: [in this window] [in a new window]   AT1 Receptor and/or Nox2 Immunoreactivities in DiA-Labeled or -Unlabeled Rat dmNTS Neurons

Ang II Increases ROS Production and Ca²⁺ Currents in dmNTS Neurons
We investigated effects of Ang II on ROS production and Ca²⁺ currents in rat dmNTS neurons. Ang II was applied at concentrations from 3 nmol/L, comparable to endogenous Ang II concentrations in brain tissues.⁴⁵ In vehicle-treated neurons, DCF fluorescence remained stable during the monitoring period (Figure 1B and 1C). Ang II dose-dependently increased DCF intensity, reflecting an increase in ROS. The effect was initially observed at 3 nmol/L (P<0.05 versus control; n=8) and reached a plateau at 100 nmol/L with an EC₅₀=38.3 nmol/L (Figure 1D). In subsequent studies we tested the effect of Ang II at concentrations of 30 to 100 nmol/L. Ang II (100 nmol/L) was also able to enhance the nifedipine-sensitive L-type Ca²⁺ current (P<0.05 versus control; n=4; Figure 2). Hydrogen peroxide (2 mmol/L), a product of the dismutation of superoxide by superoxide dismutase, also increased the L-type Ca²⁺ current in dmNTS neurons (P<0.05 versus control; n=4; Figure 2). Finally, the ROS scavenger Mn (III) porphyrin 5,10,15,20-tetrakis (benzoic acid) porphyrin manganese (III) (MnTBAP)⁴⁶ (30 µmol/L) blocked the Ang II-induced ROS production (Figure 3A). These observations indicate that nanomolar concentrations of Ang II increase ROS production and Ca²⁺ currents in dmNTS neurons.

View larger version (39K): [in this window] [in a new window]   Figure 2. Ang II and H2O2 potentiate L-type Ca2+ currents in rat DiA-labeled dmNTS neurons. A, DiA-labeled dmNTS neuron used for patch clamping. B, Left: Ca2+ currents in the presence of vehicle (C), Ang II (A), or nifedipine (N). Right: Ca2+ currents in the presence of vehicle (C), H2O2 (H), or nifedipine (N). C, Histograms illustrating effects of Ang II (left) or H2O2 (right) on the L-type Ca2+ current (n=4/group). *P<0.05 vs control; P<0.01 vs Ang II or H2O2.

View larger version (22K): [in this window] [in a new window]   Figure 3. AT1 receptors and NADPH oxidase mediate the ROS production induced by Ang II in rat dmNTS neurons. A, The Ang II-induced increase in DCF (n=8) was abolished by MnTBAP (30 µmol/L; n=9). B, Losartan (3 µmol/L; n=8) but not PD123319 (40 µmol/L; n=6) blocked DCF signals induced by Ang II. C, The Ang II-induced increase in DCF was blocked by the NADPH oxidase assembly inhibitor apocynin (1 mmol/L; n=9) or the NADPH oxidase peptide inhibitor gp91ds-tat (1 µmol/L; n=10) but not by its scrambled version (1 µmol/L; n=10). *P<0.05 vs control; **P<0.01 vs control.

AT₁ Receptors Mediate ROS Production by NADPH Oxidase in dmNTS Neurons
To determine the Ang II receptor subtype⁴⁷ responsible for the ROS production in rat dmNTS neurons, we examined the effects of the AT₁ receptor inhibitor losartan or the AT₂ receptor inhibitor PD123319.⁴⁸ Losartan (3 µmol/L) blocked the Ang II-induced increase in DCF (P>0.05 versus control; n=8), whereas PD123319 (40 µmol/L) did not (Figure 3B). Pretreatment (30 minutes) with the NADPH oxidase assembly inhibitor apocynin (1 mmol/L)⁴⁹ or the peptide inhibitor gp91ds-tat (1 µmol/L)³⁵ blocked the Ang II-induced increase in DCF (P>0.05 versus control). In contrast, the 1 µmol/L scrambled version of gp91ds-tat had no effect (Figure 3C). These data indicate that Ang II induces ROS production in dmNTS neurons via AT₁ receptors and NADPH oxidase.

Nox2 Is Critical for Ang II-Induced Ca²⁺ Currents and ROS Production in dmNTS Neurons
To obtain direct evidence that Nox2 is involved in Ang II-induced L-type Ca²⁺ current or superoxide production, we compared Ang II-induced L-type Ca²⁺ current and ROS production in dmNTS neurons isolated from WT and Nox2-null mice. Ang II significantly potentiated L-type Ca²⁺ currents of dmNTS neurons in WT mice (P<0.05 versus control; n=4) but not in Nox2-null mice (Figure 4B and 4C). Similarly, Ang II increased DCF intensity in WT mice (P<0.01 versus control; n=6) but not in Nox2-null mice (Figure 4D). These results suggest that the enhancement of both L-type Ca²⁺ currents and ROS production by Ang II depends on ROS derived from Nox2.

View larger version (35K): [in this window] [in a new window]   Figure 4. The Ang II-mediated potentiation of L-type Ca2+ currents and ROS in mouse dmNTS neurons depends on Nox2. A, DiA-labeled dmNTS neuron from a WT mouse. B, Left panel: Ca2+ currents in a WT dmNTS neuron in the presence of vehicle (C), Ang II (A) and nifedipine (N). Right panel: Ca2+ currents of in a Nox2-null mice dmNTS neuron in the presence of vehicle (C), Ang II (A), and nifedipine (N). C, Histograms showing effects of Ang II on nifedipine-sensitive L-type Ca2+ currents in dmNTS neurons from WT or Nox2-null mice (n=4). D, Effects of Ang II on DCF intensity in dmNTS neurons from WT (n=6) or Nox2-null mice (n=6). **P<0.01 vs control; P<0.01 vs Ang II.

Ang II-Elicited ROS Production Depends on Intracellular Ca²⁺ and PKC
To examine the role of intracellular Ca²⁺ in Ang II-induced ROS production, rat dmNTS neurons were treated continuously with thapsigargin (10 µmol/L) to deplete intracellular Ca²⁺ stores.⁵⁰ Pretreatment with thapsigargin (for 30 minutes) partially attenuated the increase in DCF elicited by Ang II (P<0.05 versus control; n=6). Pretreatment with thapsigargin in conjunction with removal of extracellular Ca²⁺ (for 30 minutes), however, completely blocked the Ang II-induced ROS production (P>0.05 versus control; n=7; Figure 5). To determine whether Ca²⁺ influx through voltage-gated Ca²⁺ channels plays a role in the ROS production, we used the L-type Ca²⁺ channel blocker nifedipine (5 µmol/L) or a combination of N-type Ca²⁺ channel blocker -conotoxinGVIA (600 nmol/L) and P/Q-type Ca²⁺ channel blocker AgaIVA (300 nmol/L). These inhibitors failed to alter the Ang II-mediated increase in DCF (Figure 5). We then examined the role of PKC in the Ang II-induced ROS production. Pretreatment with the PKC inhibitor GF109203X⁵¹ (15 µmol/L) abolished the Ang II-induced DCF increase (P>0.05 versus control; n=11; Figure 5). Taken together, these results suggest that intracellular Ca²⁺ and PKC are involved in the Ang II-induced, Nox2-dependent ROS production in dmNTS neurons.

View larger version (59K): [in this window] [in a new window]   Figure 5. Ca2+ and PKC play a critical role in Ang II-mediated ROS production in rat dmNTS neurons. Pretreatment with thapsigargin (10 µmol/L) attenuated the increase in DCF intensity induced by Ang II (n=6). Pretreatment with thapsigargin in Ca2+-free buffer completely blocked the Ang II-mediated increase in DCF (n=7). However, pretreatment of the L-type Ca2+ channel blocker nifedipine (5 µmol/L; n=8) or combination of the N-type Ca2+ channel blocker GVIA (600 nmol/L) and P/Q type Ca2+ channel AgaIVA (300 nmol/L; n=9) did not attenuate the Ang II-induced increase in DCF intensity. Pretreatment with the PKC inhibitor GF109203X (15 µmol/L) blocked the Ang II-mediated increase in DCF (n=11). *P<0.05 vs control; **P<0.01 vs control; P<0.01 vs nifedipine plus Ang II.

	Discussion

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There are several new findings in this study. First, using an ROS-sensitive dye, we demonstrated that Ang II, at nanomolar concentrations, elicits AT₁ receptor-dependent ROS production coupled with potentiation of Ca²⁺ currents in dmNTS neurons. The EC₅₀ of the ROS production (38.3 nmol/L) was virtually identical to that reported previously for the effects of Ang II on the L-type Ca²⁺ current (37.4 nmol/L).²⁵ Second, using Nox2-null mice, we provided the first demonstration that the Ang II-mediated increase in ROS production and related potentiation of L-type Ca²⁺ currents involve an oxidase containing Nox2 as the catalytic subunit. Third, we demonstrated that intracellular Ca²⁺ and PKC activation are critical for the ROS production evoked by Ang II in dmNTS neurons. These findings collectively provide new evidence indicating that AT₁ receptor-induced Nox2 activation and production of ROS leads to potentiation of L-type Ca²⁺ currents in dmNTS neurons, which may play a role in the vascular dysregulation mediated by central autonomic networks.

To measure ROS, we used the ROS-sensitive dye C-DCDHF-DA, which has been thoroughly tested and extensively used in studies of NADPH oxidase-dependent ROS production.^41–43 C-DCDHF-DA is oxidized by a variety of ROS, including H₂O₂, peroxynitrite, and superoxide.^41–43 The validity of the ROS detection method used in the present study is demonstrated by the observations that the DCF signal is blocked by: (1) the ROS scavengers MnTBAP, (2) a NADPH oxidase peptide inhibitor, and (3) constitutive genetic inactivation of Nox2. These observations also rule out the possibility that ROS-independent formation of DCF is attributable, for example, to auto-oxidation of C-DCDHF-DA or conversion of C-DCDHF-DA to DCF by cytochrome c.⁵²

Production of superoxide by NADPH oxidase is dependent on assembly of the cytosolic regulatory subunits with the membrane-bound subunits of the enzyme. A key step in this process is regulated by protein phosphorylation, particularly the PKC-dependent phosphorylation of p47^phox.^31–33 Our finding that a PKC inhibitor blocks Ang II-mediated ROS production in dmNTS neurons suggests that PKC, presumably by phosphorylating p47^phox, is essential for the NADPH activation induced by Ang II. We also found that the ROS production evoked by Ang II requires Ca²⁺, suggesting that the PKC involved is Ca²⁺ dependent. Concerning the sources of Ca²⁺, our findings with thapsigargin suggest that Ca²⁺ release from intracellular stores is needed for the full expression of the ROS increase. An involvement of Ca²⁺ from intracellular stores is also suggested by the study of Gebke et al,⁵³ who showed in mixed cultures of subfornical organ and organum vasculosum of the lamina terminal neurons that Ang II increases intracellular Ca²⁺ in the absence of extracellular Ca²⁺. Furthermore, Sumners et al⁵⁴ provided evidence that in hypothalamic neurons Ang II induces Ca²⁺ release from intracellular stores. Although we cannot completely rule out the role of extracellular Ca²⁺ in the ROS production, Ca²⁺ influx via voltage-gated Ca²⁺ channels does not seem to contribute to the ROS production triggered by Ang II. Ang II does activate the L-type Ca²⁺ channels, but our data suggest that this effect is mediated by Nox2-dependent ROS production.

A previous study in the Neuro-2A cell line showed that the increase in ROS produced by Ang II is not affected by removal of Ca²⁺.³⁴ This is in contrast to our findings in dmNTS neurons in which Ca²⁺ was an absolute requirement for the ROS production. The reasons for this discrepancy are not entirely clear. In addition to intrinsic differences between freshly isolated dmNTS neurons and Neuro-2A cells, the Ang II concentration is likely to be an important factor, because higher concentrations of Ang II (2 µmol/L) elicit increases in ROS in dmNTS neurons that are not blocked by Ca²⁺ removal (Supplemental Figure I, available online at http://hyper.ahajournals.org). Considering that, in the study of Zimmerman et al,³⁴ Ang II was applied at 5 µmol/L, it is conceivable that the discrepancy in the results is because of the difference in Ang II concentration used.

An important consequence of ROS production by Ang II in central autonomic neurons is the resultant increase in intracellular Ca²⁺ levels.^{25,34,40,55,56} The present data are consistent with previous reports that the elevation of intracellular Ca²⁺ induced by Ang II is secondary to the action of ROS on L-type voltage-gated Ca²⁺ channels.^25,34 However, the mechanisms underlying the effects of Nox2-derived ROS on voltage-gated Ca²⁺ influx remain unclear. One possibility is that oxidative stress modulates voltage-gated Ca²⁺ channels. Indeed, it is well established that ROS increase voltage-gated Ca²⁺ currents in neurons.⁵⁵ Our finding that H₂O₂ mimics the effect of Ang II on Ca²⁺ currents in dmNTS neurons supports this possibility, albeit indirectly.

The Ang II-induced potentiation of voltage-gated Ca²⁺ currents elicited by NADPH oxidase-dependent superoxide production is an important modulator of the excitability of dmNTS neurons.^25,39,40 The NTS is a crucial coordinator of cardiorespiratory processes and an important component of the central renin-angiotensin system.^1–5 Within the dmNTS, neurons or glia have been shown to express renin, angiotensinogen, or angiotensin-converting enzyme, as well as Angs I, II, and III and angiotensin AT₁ receptors.¹⁰ Using dual-labeling immunofluorescence electron microscopy, we demonstrated colocalization of AT₁ receptors and Nox2 in single DiA-labeled NTS neurons, indicating that a population of second-order dmNTS sensory neurons contains the AT₁ receptor and Nox2.²⁵ Thus, AT₁ receptors are located in functional surface membrane sites in Nox2-containing neurons that are contacted by vagal-like and nonvagal afferents in the dmNTS.

Long-term modulation of baroreflex function and disturbances in the renin-angiotensin system are each associated with experimental and clinical hypertension.^11,57 The mechanisms mediating adaptations in the baroreflexes presumably involve complex peripheral and central processes in which Ang II plays a role. For example, Ang II-dependent systemic hypertension is associated with increased sympathetic nerve activity,⁶ whereas Ang II-induced sympathoexcitation has also been linked to NADPH oxidase-derived ROS.^15–18 Thus, our findings suggest that Ang II-induced ROS production and L-type Ca²⁺ currents might contribute to the central autonomic effects of Ang II.^9,58,59 Moreover, considering the well-established link among L-type Ca²⁺ channels, ROS, and cellular plasticity,^60,61 heightened activation of AT₁ receptors in dmNTS neurons may also play a role in the reorganization of autonomic function accompanying hypertension.

Perspectives
The dmNTS is a critical central relay for cardiopulmonary afferents regulating cardiovascular homeostasis. We have demonstrated that Nox2 and AT₁ receptors are present in second-order dmNTS neurons in which exposure to Ang II induces ROS production and activation of Ca²⁺ currents. The Ang II-induced ROS production and Ca²⁺ currents were not observed in mice lacking Nox2. Furthermore, we have shown that Ang II-dependent ROS production requires intracellular Ca²⁺ and PKC. The data indicate that Ang II induces ROS production in second-order dmNTS neurons through a Nox2-containing NADPH oxidase. The NADPH oxidase-derived ROS, in turn, activate L-type Ca²⁺ channels. These findings provide the mechanistic basis for the powerful actions of Ang II in dmNTS and support the concept that ROS derived from Nox2 are critical signaling molecules in central autonomic neurons. They also support the growing evidence that ROS derived from Nox2 play a vital role in both central and peripheral mechanisms of hypertension.

	Acknowledgments

 
Sources of Funding

This work was supported by the National Institutes of Health grant HL18974.

Disclosures

None.

Received May 30, 2006; first decision June 15, 2006; accepted July 6, 2006.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Reis DJ. The brain and hypertension: reflections on 35 years of inquiry into the neurobiology of the circulation. Circulation. 1984; 70: 11131–11145.

2. Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev. 1994; 74: 323–364.[Free Full Text]

3. Aicher SA, Goldberg A, Sharma S, Pickel VM. µ-Opioid receptors are present in vagal afferents and their dendritic targets in the medial nucleus tractus solitarius. J Comp Neurol. 2000; 422: 181–190.[CrossRef][Medline] [Order article via Infotrieve]

4. Mifflin SW. What does the brain know about blood pressure? News Physiol Sci. 2001; 16: 266–271.[Abstract/Free Full Text]

5. Boscan P, Pickering AE, Paton JF. The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents. Exp Physiol. 2002; 87: 259–266.[Abstract]

6. McCubbin JW, DeMoura RS, Page IH, Olmsted F. Arterial hypertension elicited by subpressor amounts of angiotensin. Science. 1965; 49: 1394–1395.

7. Hogarty DC, Speakman EA, Puig V, Phillips MI. The role of angiotensin, AT₁ and AT₂ receptors in the pressor, drinking and vasopressin responses to central angiotensin. Brain Res. 1992; 586: 289–294.[CrossRef][Medline] [Order article via Infotrieve]

8. Phillips MI, Sumners C. Angiotensin II in central nervous system physiology. Regul Pept. 1998; 78: 1–11.[CrossRef][Medline] [Order article via Infotrieve]

9. Ferguson AV, Washburn DL, Latchford KJ. Hormonal and neurotransmitter roles for angiotensin in the regulation of central autonomic function. Exp Biol Med. 2001; 226: 85–96.[Abstract/Free Full Text]

10. Diz DI, Jessup JA, Westwood BM, Bosch SM, Vinsant S, Gallengher PE, Averill DB. Angiotensin peptides as neurotransmitters/neuromodulators in the dorsomedial medulla. Clin Exp Pharmacol Physiol. 2002; 29: 473–482.[CrossRef][Medline] [Order article via Infotrieve]

11. Lohmeier TE, Lohmeier JR, Warren S, May PJ, Cunningham JT. Sustained activation of the central baroreceptor pathway in angiotensin hypertension. Hypertension. 2002; 39: 550–556.[Abstract/Free Full Text]

12. Huang J, Hara Y, Anrather J, Speth RC, Iadecola C, Pickel VM. Angiotensin II subtype 1A (AT_1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin. Neuroscience. 2003; 122: 21–36.[CrossRef][Medline] [Order article via Infotrieve]

13. Glass MJ, Huang J, Speth RC, Iadecola C, Pickel VM. Angiotensin II AT-1A receptor immunolabeling in rat medial nucleus tractus solitarius neurons: subcellular targeting and relationships with catecholamines. Neuroscience. 2005; 130: 713–723.[CrossRef][Medline] [Order article via Infotrieve]

14. Paton JFR, Kasparov S. Sensory channel specific modulation in the nucleus of the solitary tract. J Auto Nerves Syst. 2000; 80: 117–129.

15. Lindley TE, Doobay MF, Sharma RV, Davisson RL. Superoxide is involved in the central nervous system activation and sympathoexcitation of myocardial infarction-induced heart failure. Circ Res. 2004; 94: 402–409.[Abstract/Free Full Text]

16. Zimmerman MC, Lazartigues E, Sharma RV, Davisson RL. Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervous system. Circ Res. 2004; 95: 210–216.[Abstract/Free Full Text]

17. Zucker IH. Brain angiotensin II: new insights into its role in sympathetic regulation. Circ Res. 2002; 90: 503–505.[Free Full Text]

18. Gao L, Wang W, Li YL, Schultz HD, Liu D, Cornish KG, Zucker IH. Superoxide mediates sympathoexcitation in heart failure: roles of angiotensin II and NAD(P)H oxidase. Circ Res. 2004; 95: 937–944.[Abstract/Free Full Text]

19. Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev. 2001; 52: 11–34.

20. Sumners C, Zhu M, Gelband CH, Posner P. Angiotensin II type 1 receptor modulation of neuronal K⁺ and Ca²⁺ currents: intracellular mechanisms. Am J Physiol Cell Physiol. 1996; 271: C154–C163.[Abstract/Free Full Text]

21. Sun C, Sellers KW, Sumners C, Raizada MK. NAD(P)H oxidase inhibition attenuates neuronal chronotropic actions of angiotensin II. Circ Res. 2005; 96: 659–666.[Abstract/Free Full Text]

22. Lassègue B, Clempus RE. Vascular NAD(P)H oxidases specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277–R297.[Abstract/Free Full Text]

23. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nature Rev Immunol. 2004; 4: 181–189.[CrossRef][Medline] [Order article via Infotrieve]

24. Serrano F, Klann E. Reactive oxygen species and synaptic plasticity in aging hippocampus. Ageing Res Rev. 2004; 3: 431–443.[CrossRef][Medline] [Order article via Infotrieve]

25. Wang G, Anrather J, Huang J, Speth RC, Pickel VM, Iadecola C. NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius. J Neurosci. 2004; 24: 5516–5524.[Abstract/Free Full Text]

26. Kazama K, Anrather J, Zhou P, Girouard H, Frys K, Milner TA, Iadecola C. Angiotensin II impairs neurovascular coupling in neocortex through NADPH-oxidase-derived radicals. Circ Res. 2004; 95: 1019–1026.[Abstract/Free Full Text]

27. Pollock JD, Williams DA, Gifford MA, Li LL, Du X, Fisherman J, Orkin SH, Doerschuk CM, Dinauer MC. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet. 1995; 9: 202–209.[CrossRef][Medline] [Order article via Infotrieve]

28. Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular disease. Trends Pharmacol Sci. 2003; 24: 471–478.[CrossRef][Medline] [Order article via Infotrieve]

29. Touyz RM. Reactive oxygen species as mediators of calcium signaling by angiotensin II: implications in vascular physiology and pathophysiology. Antioxid Redox Signal. 2005; 7: 1302–1314.[CrossRef][Medline] [Order article via Infotrieve]

30. Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res. 2006; 98: 453–462.[Abstract/Free Full Text]

31. Hoyal CR, Gutierrez A, Young BM, Catz SD, Lin JH, Tsichlis PN, Babior BM. Modulation of p47^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase. Proc Natl Acad Sci USA. 2003; 100: 5130–5135.[Abstract/Free Full Text]

32. Faust LR, el Benna J, Babior BM, Chanock SJ. The phosphorylation targets of p47^phox, a subunit of the respiratory burst oxidase, functions of the individual target serines as evaluated by site-directed mutagenesis. J Clin Invest. 1995; 96: 1499–1505.[Medline] [Order article via Infotrieve]

33. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]

34. Zimmerman MC, Sharma RV, Davisson RL. Superoxide mediates angiotensin II-induced influx of extracellular calcium in neural cells. Hypertension. 2005; 45: 717–723.[Abstract/Free Full Text]

35. Rey FE, Cifuentes ME, Kiarash A, Quinn MT, Pagano PJ. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O₂^– and systolic blood pressure in mice. Circ Res. 2001; 89: 408–414.[Abstract/Free Full Text]

36. Mendelowitz D, Yang M, Andresen MC, Kunze DL. Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminals on neurons from the nucleus tractus solitarius. Brain Res. 1992; 581: 339–343.[CrossRef][Medline] [Order article via Infotrieve]

37. Balkowiec A, Kunze DL, Katz DM. Brain-derived neurotrophic factor acutely inhibits AMPA-mediated currents in developing sensory relay neurons. J Neurosci. 2000; 20: 1904–1911.[Abstract/Free Full Text]

38. Glaum SR, Miller RJ. Activation of metabotropic glutamate receptors produces reciprocal regulation of ionotropic glutamate and GABA responses in the nucleus of the tractus solitarius of the rat. J Neurosci. 1993; 13: 1636–1641.[Abstract]

39. Ishibashi H, Akaike N. Norepinephrine modulates high voltage-activated calcium channels in freshly dissociated rat nucleus tractus solitarii neurons. Neuroscience. 1995; 68: 1139–1146.[CrossRef][Medline] [Order article via Infotrieve]

40. Endoh T. Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II. J Physiol (Lond). 2005; 568: 851–856.[Abstract/Free Full Text]

41. Hempel SL, Buettner GR, O’Malley YQ, Wessels DA, Flaherty DM. Dihydrofluo-rescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichloro-dihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med. 1999; 27: 146–159.[CrossRef][Medline] [Order article via Infotrieve]

42. Miller FJ Jr, Griendling KK. Functional evaluation of nonphagocytic NAD(P)H oxidases. Methods Enzymol. 2002; 353: 220–233.[Medline] [Order article via Infotrieve]

43. Keller A, Mohamed A, Drose S, Brandt U, Fleming I, Brandes RP. Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. Free Radic Res. 2004; 38: 1257–1267.[CrossRef][Medline] [Order article via Infotrieve]

44. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 1981; 391: 85–100.[CrossRef][Medline] [Order article via Infotrieve]

45. Zhao X, White R, Huang BS, Huysse VJ, Leenen FHH. High salt intake and the rennin-angiotensin system in Dahl salt-sensitive rats. J Hyperten. 2001; 19: 89–98.[CrossRef][Medline] [Order article via Infotrieve]

46. Patel M, Day BJ. Metalloporphyrin class of therapeutic catalytic antioxidants. Trends Pharmacol Sci. 1999; 20: 359–364.[CrossRef][Medline] [Order article via Infotrieve]

47. Lenkei Z, Palkovits M, Corvol P, Llorens-Cortes C. Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs in the adult rat brain: a functional neuroanatomical review. Front Neuroendocrinol. 1997; 18: 383–439.[CrossRef][Medline] [Order article via Infotrieve]

48. Timmermans PB, Smith RD. The diversified pharmacology of angiotensin II-receptor blockade. Blood Press. 1996; 2 (suppl): 53–61.

49. Suzuki Y, Wang W, Vu TH, Raffin TA. Effect of NADPH oxidase inhibition on endothelial cell ELAM-1 mRNA expression. Biochem Biophys Res Commun. 1992; 184: 1339–1343.[CrossRef][Medline] [Order article via Infotrieve]

50. Treiman M, Caspersen C, Christensen SB. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPases. Trends Pharmacol Sci. 1998; 19: 131–135.[CrossRef][Medline] [Order article via Infotrieve]

51. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991; 266: 15771–15781.[Abstract/Free Full Text]

52. Burkitt MJ, Wardman P. Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis. Biochem Biophys Res Commun. 2001; 282: 329–333.[CrossRef][Medline] [Order article via Infotrieve]

53. Gebke E, Muller AR, Jurzak M, Gerstberger R. Angiotensin II-induced calcium signalling in neurons and astrocytes of rat circumventricular organs. Neuroscience. 1998; 85: 509–520.[CrossRef][Medline] [Order article via Infotrieve]

54. Sumners C, Zhu M, Gelband CH, Posner P. Angiotensin II type 1 receptor modulation of neuronal K⁺ and Ca²⁺ currents: intracellular mechanisms. Am J Physiol Cell Physiol. 1996; 271: C154–C163.[Abstract/Free Full Text]

55. Li A, Segui J, Heinemann SH, Hoshi T. Oxidation regulates cloned neuronal voltage-dependent Ca²⁺ channels expressed in Xenopus oocytes. J Neurosci. 1998; 18: 6740–6747.[Abstract/Free Full Text]

56. Washburn DL, Ferguson AV. Selective potentiation of N-type calcium channels by angiotensin II in rat subfornical organ neurones. J Physiol. 2001; 536: 667–675.[Abstract/Free Full Text]

57. Davisson RL. Physiological genomic analysis of the brain renin-angiotensin system. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R498–R511.[Abstract/Free Full Text]

58. Kasparov S, Paton JFR. Differential effects of angiotensin II in the nucleus tractus solitarii of the rat-plausible neuronal mechanisms. J Phyisol (Lond). 1999; 521: 227–238.

59. Zimmerman MC, Lazartigues E, Lang JA, Sinnayah P, Ahmad IM, Spitz DR, Davisson RL. Superoxide mediates the actions of angiotensin II in the central nervous system. Circ Res. 2002; 91: 1038–1045.[Abstract/Free Full Text]

60. Wright JW, Reichert JR, Davis CJ, Harding JW. Neural plasticity and the brain renin-angiotensin system. Neurosci Biobehav Rev. 2002; 26: 529–552.[CrossRef][Medline] [Order article via Infotrieve]

61. Morgan SL, Teyler TJ. VDCCs and NMDARs underlie two forms of LTP in CA1 hippocampus in vivo. J Neurophysiol. 1999; 82: 736–740.[Abstract/Free Full Text]

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