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(Hypertension. 2005;45:717.)
© 2005 American Heart Association, Inc.

Original Articles

Superoxide Mediates Angiotensin II–Induced Influx of Extracellular Calcium in Neural Cells

From the Department of Anatomy and Cell Biology (M.C.Z., R.V.S., R.L.D.); Free Radical and Radiation Biology Program, Department of Radiation Oncology (M.C.Z., R.L.D.); and The Cardiovascular Center (R.V.S., R.L.D.), The University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City.

Correspondence to Robin L. Davisson, PhD, Department of Anatomy and Cell Biology, 1-418 Bowen Science Building, The University of Iowa College of Medicine, Iowa City, IA 52242. E-mail robin-davisson{at}uiowa.edu

	Abstract

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We recently demonstrated that superoxide (O₂^•–) is a key signaling intermediate in central angiotensin II (Ang II)-elicited blood pressure and drinking responses, and that hypertension caused by systemic Ang II infusion involves oxidative stress in cardiovascular nuclei of the brain. Intracellular Ca²⁺ is known to play an important role in Ang II signaling in neurons, and it is also linked to reactive oxygen species mechanisms in neurons and other cell types. However, the potential cross-talk between Ang II, O₂^•–, and Ca²⁺ in neural cells remains unknown. Using mouse neuroblastoma Neuro-2A cells, we tested the hypothesis that O₂^•– radicals are involved in the Ang II–induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Ang II caused a rapid time-dependent increase in [Ca²⁺]_i that was abolished in cells bathed in Ca²⁺-free medium or by pretreatment with the nonspecific voltage-gated Ca²⁺ channel blocker CdCl₂, suggesting that voltage-sensitive Ca²⁺ channels are the primary source of Ang II–induced increases in [Ca²⁺]_i in this cell type. Overexpression of cytoplasm-targeted O₂^•– dismutase via an adenoviral vector (AdCuZnSOD) efficiently scavenged Ang II–induced increases in intracellular O₂^•– and markedly attenuated the increase in [Ca²⁺]_i caused by this peptide. Furthermore, adenoviral-mediated expression of a dominant-negative isoform of Rac1 (AdN17Rac1), a critical component for NADPH oxidase activation and O₂^•– production, significantly inhibited the increase in [Ca²⁺]_i after Ang II stimulation. These data provide the first evidence that O₂^•– is involved in the Ang II–stimulated influx of extracellular Ca²⁺ in neural cells and suggest a potential intracellular signaling mechanism involved in Ang II–mediated oxidant regulation of central neural control of blood pressure.

Key Words: calcium channels • oxidative stress • imaging • central nervous system • renin-angiotensin system

	Introduction

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The best-known physiological effect of angiotensin II (Ang II) acting in the central nervous system (CNS) is modulation of body fluid and cardiovascular homeostasis. Ang II, acting primarily via Ang II type-1 (AT₁) receptors located in central cardiovascular control regions, causes increases in blood pressure,¹ water intake,² vasopressin release,³ and sympathoexcitation.¹ Dysregulation of central angiotensinergic signaling mechanisms is associated with numerous pathological cardiovascular conditions.⁴ Therefore, elucidating the intracellular signaling mechanisms by which Ang II modulates neuronal activity is critical to our understanding of central Ang II–dependent cardiovascular diseases such as hypertension and heart failure.

We have recently identified reactive oxygen species (ROS), particularly superoxide anions (O₂^•–), as key signaling intermediates in Ang II–stimulated activation of CNS neurons. Increased scavenging of cytoplasmic O₂^•– in key cardiovascular regulatory nuclei in the brain causes marked attenuation of the pressor effects elicited by Ang II administered either directly into the CNS⁵ or via the systemic circulation.⁶ More recently, our studies have shown that a Rac1-activated NADPH oxidase complex is the primary source of Ang II–induced O₂^•– production in neurons.⁷ Although these studies have identified an important functional role of O₂^•– in central Ang II–mediated cardiovascular responses, the signaling mechanisms of O₂^•– in Ang II–mediated neuronal activation remain to be identified.

A potential downstream target of Ang II–induced O₂^•– production may involve Ca²⁺ signaling. It is well established that Ang II stimulates an increase in total Ca²⁺ current and a decrease in total K⁺ current,⁸ which leads to an increase in neuronal firing rate by controlling action potential generation.^9,10 Interestingly, in neurons, it has been shown that ROS induce an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that has been linked to cellular toxicity and neurodegenerative diseases. In fact, it is postulated that ROS may alter numerous proteins involved in Ca²⁺ signaling and neuronal firing, including voltage-gated K⁺ and Ca²⁺ channels.^11–13 Whereas these studies have provided important evidence for a signaling cascade that involve both ROS and Ca²⁺ in neurotoxicity, the cross-talk between these two important intermediates in Ang II signaling mechanisms involved in the central regulation of cardiovascular function remains to be investigated.

The goal of this study was to address the hypothesis that Ang II–stimulated increases in neuronal [Ca²⁺]_i involves ROS generation. Using the mouse neuroblastoma cell line Neuro-2A, recently shown to express high levels of AT₁ receptors,¹⁴ we modulated O₂^•– levels with adenoviral vectors encoding either the cytoplasm-localized superoxide dismutase (AdCuZnSOD) or a dominant-negative isoform of an essential component of NADPH oxidase activation, Rac1 (AdN17Rac1). Measuring Ang II–induced increases in [Ca²⁺]_i via Fura-2 ratio fluorescence imaging, our results show that Ang II–mediated Ca²⁺ influx through voltage-sensitive Ca²⁺ channels involves NADPH oxidase-derived O₂^•– production.

	Materials and Methods

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Adenoviral Vectors and Gene Transfer
Neuro-2A cells obtained from ATCC (Manassas, Va) were grown on 25-mm coverslips, as described previously.⁷ Cells were infected with recombinant adenoviral vectors encoding ß-galactosidase (AdLacZ), cytoplasm-targeted superoxide dismutase (AdCuZnSOD), or the dominant-negative mutant of Rac1 (AdN17Rac1) 24 hours before experimentation. Each of these viral vectors were constructed and characterized as described in detail previously.^7,15

[Ca²⁺]_i Measurements
Ang II–stimulated changes in intracellular calcium concentration ([Ca²⁺]_i) were assessed by Fura-2 fluorescence ratio imaging using a microscopic digital imaging system (Photon Technology International) as described previously.^16–18 Briefly, subconfluent Neuro-2A cells grown on 25-mm coverslips were loaded with the Ca²⁺-specific dye Fura-2 by incubating with 1 µmol/L Fura-2AM (Molecular Probes) at 37°C for 30 minutes. To examine the role of AT₁ receptors, Neuro2A cells were treated with the AT₁ receptor antagonist losartan (10 µmol/L) for 30 minutes before Ang II stimulation. Next, to determine the role of extracellular Ca²⁺ influx in the Ang II–stimulated increase in [Ca²⁺]_i, separate subsets of cells were bathed in Ca²⁺-free Hank’s balanced salt solution containing the Ca²⁺ chelator EGTA (2 mmol/L) for 2 to 5 minutes or in normal Ca²⁺-containing Hank’s balanced salt solution containing CaCl₂ (1.26 mmol/L) and stimulated with Ang II prepared in the respective medium. Additionally, separate cultures were pretreated with the nonspecific voltage-sensitive Ca²⁺ channel blocker CdCl₂ (125 µmol/L; Fisher Scientific, New Lawn, NJ) for 5 minutes before Ang II stimulation to block Ca²⁺ influx from voltage-gated Ca²⁺ channels. To determine the patency of intracellular Ca²⁺ stores in cells incubated in Ca²⁺-free medium or pretreated with CdCl₂, cultures were treated with thapsigargin (5 µg/mL; Sigma-Aldrich Co, St. Louis, Mo) after Ang II stimulation. Thapsigargin is known to release Ca²⁺ from the endoplasmic reticulum by inhibiting Ca²⁺ ATPase.¹⁹ Finally, to examine the role of intracellular O₂^•– in Ang II–stimulated increases in [Ca²⁺]_i in neurons, we targeted O₂^•– levels by two different modes. First, to scavenge cytosolic O₂^•–, Neuro-2A cells were infected with AdCuZnSOD (100 pfu/cell) 24 hours before Ang II stimulation and [Ca²⁺]_i measurements. In separate cultures, cells were transduced 24 hours before Ang II treatment with AdN17Rac1 (100 pfu/cell) to inhibit Rac1-mediated NADPH oxidase assembly, activation, and subsequent O₂^•– production. In both experiments, to control for possible effects of the adenoviral vector itself, cells were treated with AdLacZ (100 pfu/cell).

Detection of O₂^•– Generation
Changes in O₂^•– generation in Neuro-2A cells after Ang II (5 µmol/L) were measured using the fluorogenic probe dihydroethidium (DHE; 5 µmol/L, Molecular Probes, Inc) as described.⁷ To confirm the fidelity of the assay, separate cultures were transfected with AdCuZnSOD (100 pfu/cell) 24 hours before DHE loading.

Statistics
Data were analyzed by Student t test when comparing only 2 groups and by ANOVA, followed by Newman–Keuls correction for multiple comparisons when comparing >2 groups. Data were expressed as mean±SEM and differences were considered significant at P<0.05.

	Results

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Ang II Stimulates an Increase in [Ca²⁺]_i in Neuro-2A Cells
A number of studies have shown that Ang II produces an increase in [Ca²⁺]_i in primary neurons cultured from the CNS.^8,16,17,20 Our initial experiments were designed to confirm this effect of Ang II in Neuro-2A cells. The rationale for choosing this cell line was 2-fold. First, these cells have been shown to express high levels of AT₁ receptors through rigorous expression quantification methods.¹⁴ Second, our recent studies showed that Ang II induces an AT₁ receptor-dependent increase in O₂^•– generation in this cell line via NADPH oxidase activation.⁷ Data presented in Figure 1A show the Ang II–elicited response of all cells in the field of view from a representative coverslip. Similar to the response described in primary neurons cultured from the brain,^16,17,20 Ang II caused a time-dependent increase in [Ca²⁺]_i in Neuro-2A cells, which peaked between 20 to 30 seconds after stimulation and then returned to near basal levels by 60 seconds. This response was virtually abolished in cells pretreated with losartan (Figure 1A), suggesting that similar to primary neurons, the Ang II–induced increase in [Ca²⁺]_i in Neuro-2A cells is mediated by the AT₁ receptor. Summary data given in Figure 1B show that Ang II caused 3-fold increase in [Ca²⁺]_i (n=90 cells from 4 coverslips) in vehicle-treated cells. This response was significantly attenuated in cells pretreated with losartan (n=124 cells from 5 coverslips; P<0.05 versus Ang II alone). It should be noted that although basal [Ca²⁺]_i appears to be slightly different between the representative coverslips shown in Figure 1A, the average basal [Ca²⁺]_i from the 90 and 124 cells analyzed was similar in both groups of cells (50±4 nmol/L in vehicle-treated cells; 51±2 in losartan-treated cells, P>0.05, Figure 1B).

View larger version (28K): [in this window] [in a new window]   Figure 1. Ang II stimulates an increase in [Ca2+]i in Neuro-2A cells. A, Representative pseudocolor 340/380-nm ratio images of Fura-2 fluorescence with computed [Ca2+]i from coverslips pretreated for 30 minutes with either vehicle (n=29 cells) or losartan (10 µmol/L, n=17 cells) before (baseline) and 20 seconds after Ang II stimulation. The blue in the scale bar indicates low levels of [Ca2+]i, whereas the red depicts high levels of [Ca2+]i. B, Summary data (mean±SEM) of the basal and peak increase in [Ca2+]i in cells stimulated with Ang II after 30 minutes of pretreatment with vehicle (n=90 cells from 4 coverslips) or losartan (n=124 cells from 5 coverslips). [Ca2+]i values over the entire cell were averaged to obtain the changes in the whole cell Ca2+ concentration. *P<0.05 vs baseline, P<0.05 vs vehicle plus Ang II.

Ang II Increases Cytosolic Ca²⁺ by Stimulating an Influx of Extracellular Ca²⁺
Although numerous studies have demonstrated that Ang II causes an increase in [Ca²⁺]_i in neurons cultured from the central nervous system, the Ca²⁺ pools that are mobilized by Ang II appear to vary.^8,16,17,20 To determine the source of [Ca²⁺]_i in Ang II–stimulated Neuro-2A cells, cells were bathed either in Ca²⁺-free medium containing 2 mmol/L EGTA or in normal Ca²⁺-containing medium. Similar to the data shown in Figure 1, Ang II caused a robust time-dependent increase in [Ca²⁺]_i between 20 and 30 seconds after incubation in Ca²⁺-containing medium (n=25 cells from 1 representative coverslip; Figure 2A). This response was completely abolished in cells bathed in Ca²⁺-free medium (n=37 cells from 1 representative coverslip; Figure 2A), suggesting that the Ang II–induced increase in [Ca²⁺]_i is caused by an influx of extracellular Ca²⁺. To confirm that intracellular Ca²⁺ stores remained intact in cells bathed in Ca²⁺-free medium, cells were treated with thapsigargin to release Ca²⁺ from internal stores after Ang II stimulation. Regardless of bathing medium, thapsigargin caused a similar increase in [Ca²⁺]_i (Figure 2A), demonstrating that Neuro-2A cells bathed in Ca²⁺-free medium retain functional intracellular Ca²⁺ stores. Data summarized from 4 to 6 separate coverslips show that Ca²⁺-free medium did not alter either the basal Ca²⁺ values or the thapsigargin-mediated increase in [Ca²⁺]_i (Figure 2B). In contrast, the Ang II–stimulated increase in peak [Ca²⁺]_i was completely abolished in Ca²⁺-free medium compared with Ca²⁺-containing medium (Figure 2B).

View larger version (24K): [in this window] [in a new window]   Figure 2. Ang II–stimulated increase in [Ca2+]i in Neuro-2A cells is dependent on extracellular Ca2+. A, Average data from one representative coverslip in each treatment group demonstrating the changes in [Ca2+]i vs time in normal Ca2+-containing medium (n=25 cells) or Ca2+-free medium containing 2 mmol/L EGTA (n=37 cells) in response to Ang II stimulation. After [Ca2+]i returned to baseline, cultures were treated with thapsigargin to confirm the functional integrity of intracellular Ca2+ stores in both treatment groups. B, Summary data (mean±SEM) of the basal and the peak increase in [Ca2+]i after Ang II or thapsigargin stimulation in cells bathed in Ca2+-containing (n=80 cells from 4 coverslips) or Ca2+-free medium (n=138 cells from 6 coverslips). *P<0.05 vs baseline; P<0.05 vs Ca2+-containing medium plus Ang II.

Ang II–Induced Influx of Extracellular Ca²⁺ Involves Voltage-Sensitive Channels
Although data presented in Figure 2 demonstrate that the Ang II–induced increase in [Ca²⁺]_i is extracellular Ca²⁺-dependent, we sought to extend these studies and examine the role of voltage-gated Ca²⁺ channels in the Ang II–induced Ca²⁺ influx. It has been shown in neurons that Ang II–activated Ca²⁺ channels are inhibited by the nonspecific voltage-sensitive Ca²⁺ channel blocker Cd²⁺.⁹ As such, cultures were pretreated for 5 minutes with CdCl₂ (125 µmol/L) before Ang II stimulation and [Ca²⁺]_i measurements. Summary data presented in Figure 3 show that the mean peak Ang II–induced increase in [Ca²⁺]_i was markedly attenuated by pretreatment with CdCl₂ (n=148 cells on 5 coverslips) compared with vehicle-treated cells (n=59 cells on 3 coverslips; P<0.05). These data suggest that Ang II modulates voltage-sensitive Ca²⁺ channels, resulting in an influx of extracellular Ca²⁺ into Neuro-2A cells. To verify that CdCl₂ pretreatment does not affect internal Ca²⁺ stores, vehicle-treated or CdCl₂-treated cells were stimulated with thapsigargin after Ang II stimulation. Thapsigargin induced comparable increases in [Ca²⁺]_i in both groups of cells (Figure 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Ang II–stimulated influx of extracellular Ca2+ involves voltage-sensitive Ca2+ channels. Cells were treated with vehicle or CdCl2 (125 µmol/L) for 5 minutes before Ang II stimulation. After the resolution of the Ang II response, cells were treated with thapsigargin to verify the integrity of intracellular Ca2+ stores in both groups. Summary data (mean±SEM) of the baseline [Ca2+]i and the peak increase in [Ca2+]i after Ang II or thapsigargin stimulation in cells pretreated with vehicle (n=59 cells on 3 coverslips) or CdCl2 (n=148 cells on 5 coverslips). *P<0.05 vs baseline; P<0.05 vs vehicle plus Ang II.

Superoxide Mediates Ang II–Induced Influx of Ca²⁺ in Neuro-2A Cells
We have shown in primary neurons and in Neuro-2A cells that Ang II stimulates an increase in O₂^•– production.^5,7 Furthermore, a Rac1-activated NADPH oxidase plays an important role in this response in vitro, and is implicated in the in vivo effects of central Ang II on blood pressure.⁷ To investigate whether there is a functional link between Ang II–stimulated increases in O₂^•– and [Ca²⁺]_i in neurons, Neuro-2A cells were infected with AdCuZnSOD to increase O₂^•– scavenging or AdN17Rac1 to inhibit NADPH oxidase activation and subsequent O₂^•– production. Overexpression of CuZnSOD caused a significant blunting of the Ang II–induced increase in [Ca²⁺]_i as seen in the tracings of all cells from one representative coverslip in Figure 4A (n=40 cells). Similarly, inhibition of NADPH oxidase activation by adenoviral expression of N17Rac1 nearly abolished Ang II–induced increases in [Ca²⁺]_i (n=29 cells on 1 coverslip; Figure 4A). Importantly, adenoviral infection itself did not affect the Ang II response, because AdLacZ-treated cells exhibited the same robust time-dependent increase in [Ca²⁺]_i (n=21 cells on 1 coverslip; Figure 4A), as seen in noninfected cells (Figure 1). Summary data in Figure 4B show that the peak Ang II–induced increase in [Ca²⁺]_i in control vector-infected cells (n=138 cells from 5 coverslips) was markedly attenuated in both AdCuZnSOD-infected (n=146 cells from 5 coverslips; P<0.05 versus AdLacZ) and AdN17Rac1-infected cells (n=136 cells from 5 coverslips; P<0.05 versus AdLacZ). These data suggest that O₂^•– plays a key signaling role in Ang II–induced increases in [Ca²⁺]_i in Neuro-2A cells.

View larger version (31K): [in this window] [in a new window]   Figure 4. Superoxide is required for Ang II–stimulated influx of extracellular Ca2+ in Neuro-2A cells. A, Representative tracings of the changes in [Ca2+]i vs time of all cells from one representative coverslip infected with AdLacZ (n=21), AdCuZnSOD (n=40), or AdN17Rac (n=29) 24 hours before Ang II stimulation. Each line represents the response of a single cell on the coverslip. Arrows indicate time of Ang II stimulation. B, Summary data (mean±SEM) showing baseline [Ca2+]i and the peak increase in [Ca2+]i after Ang II stimulation in cultures treated with AdLacZ (n=138 cells from 5 coverslips), AdCuZnSOD (n=146 cells from 5 coverslips), or AdN17Rac1 (n=136 cells from 5 coverslips) 24 hours earlier. *P<0.05 vs baseline; P<0.05 vs AdLacZ plus Ang II.

Ang II–Induced O₂^•– Production Is Not Dependent on an Increase in [Ca²⁺]_i
Although data presented in Figure 4 suggest that Ang II–induced O₂^•– production stimulates an increase in [Ca²⁺]_i in Neuro-2A cells, previous studies in other cell types have reported that an increase in [Ca²⁺]_i leads to the production of ROS.^21,22 To determine the role of Ca²⁺ in O₂^•– generation in neurons stimulated with Ang II, and to investigate a potential feed-forward loop of O₂^•–-induced Ca²⁺ and Ca²⁺-induced O₂^•– production, we measured O₂^•– generation in Ang II–stimulated Neuro-2A cells using the DHE. As we have shown previously,⁷ Ang II caused a significant time-dependent increase in DHE fluorescence in cells bathed in normal Ca²⁺-containing medium (Figure 5). Interestingly, despite complete abolition of Ang II–induced increases in [Ca²⁺]_i by Ca²⁺-free medium, cells bathed in this medium showed a similar increase in O₂^•– formation as cells bathed in normal medium. These data suggest that Ang II–stimulated increases in O₂^•– production in Neuro-2A cells are independent of changes in cytosolic Ca²⁺. It should be noted that Ang II–induced increases in DHE fluorescence were significantly inhibited in cells pretreated with losartan, as we have previously reported,⁷ and in cells infected with AdCuZnSOD 24 hours earlier (Figure 5), thus corroborating the fidelity of the assay for measuring O₂^•– levels. In addition, there was no increase in DHE fluorescence over 30 minutes in vehicle-treated cells⁷ (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 5. Ang II–induced O2•– production in Neuro-2A cells is independent of changes in [Ca2+]i. A, Representative confocal images of DHE-loaded Neuro-2A cells, showing the effects of Ang II treatment after 5 and 30 minutes in cells bathed in normal Ca2+-containing or Ca2+-free medium. To demonstrate the specificity of DHE for O2•– production, separate cultures were infected with AdCuZnSOD 24 hours before Ang II stimulation. B, Summary data of DHE fluorescence per cell before Ang II stimulation or after 5 and 30 minutes of Ang II treatment. Cells were bathed in Ca2+-containing (n=142 cells, 5 plates) or Ca2+-free medium (n=155 cells, 6 plates) during Ang II stimulation. As a control, additional cultures were treated with AdCuZnSOD (n=168 cells, 5 plates). Data are mean±SEM and expressed relative to DHE fluorescence before Ang II. *P<0.05 vs before Ang II; P<0.05 vs Ca2+-containing and Ca2+-free medium at 5 or 30 minutes.

	Discussion

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We have previously identified O₂^•– as a novel signaling intermediate in the actions of Ang II in CNS neurons.^5–7 Given the evidence that increased [Ca²⁺]_i plays a critical role in Ang II–stimulated neuronal activation,²³ we examined the cross-talk between O₂^•– and Ca²⁺ in Neuro-2A cells stimulated with Ang II. Our results demonstrate that in this cell line, similar to what is seen in primary neurons from central cardiovascular regions, Ang II increases [Ca²⁺]_i by inducing an influx of extracellular Ca²⁺ through Cd²⁺-sensitive Ca²⁺ channels. This response was markedly attenuated by gene transfer of either cytoplasm-targeted SOD or a dominant-negative isoform of Rac1 (AdN17Rac), an obligatory subunit for NADPH oxidase complex activation and O₂^•– production. These data provide the first evidence that the Ang II–induced influx of extracellular Ca²⁺ in neuronal cells is mediated by increased O₂^•– anion formation. A further important finding is that the increase in O₂^•– production is independent of the associated Ca²⁺ entry. Together, these data suggest that O₂^•– -induced modulation of Ca²⁺ influx is an important factor in Ang II signaling in neurons.

Our initial studies characterizing the Ang II–stimulated Ca²⁺ response in Neuro-2A cells were designed to establish the feasibility of using this cell line to study the interplay between Ang II, Ca²⁺, and ROS in neurons. It has been shown by a number of investigators that Ang II stimulates an increase in [Ca²⁺]_i in primary neurons derived from key central cardiovascular control regions,⁸ including the subfornical organ¹⁷ and area postrema.¹⁶ For example, Sumners et al have shown that Ang II stimulation of hypothalamic neurons leads to inositol 1,4,5-triphosphate generation and the subsequent increase in [Ca²⁺]_i.⁸ Gebke et al demonstrated that in neurons cultured from the subfornical organ or organum vasculosum of the lamina terminalis the Ang II–stimulated increase in [Ca²⁺]_i is dependent on the presence of extracellular Ca²⁺.²⁰ In support of these findings, Sumners et al reported that Ang II–induced activation of neurons cultured from the hypothalamus and brain stem is, at least in part, caused by the stimulation of Cd²⁺-sensitive voltage-gated Ca²⁺ channels.^8,9 These studies have been further extended with the identification of N-type Ca²⁺ channels as the primary voltage-sensitive Ca²⁺ channel activated by Ang II in subfornical organ neurons.²⁴ Thus, there are striking similarities in the Ang II/Ca²⁺ mechanisms between primary neurons cultured from the CNS and the mouse neuroblastoma Neuro-2A cell line. This was important because our previous studies showing Ang II–stimulated ROS generation in neurons used mouse models, and we wanted to remain with this species for the current study. Although we have cultured primary neurons from cardiovascular control regions of mouse brain and we have shown Ang II–induced O₂^•– generation,⁵ the limited number of cells obtained make this a difficult model system to employ for Ca²⁺ imaging studies. Moreover, the ease and efficiency of gene transfer is greater in this cell line compared with primary neurons. Therefore, Neuro-2A cells provide a good model for studying the role of O₂^•– in Ang II–induced Ca²⁺ responses.

Our previous evidence suggests that NADPH oxidase-derived intracellular O₂^•– anion generation in central cardiovascular sites is required for the blood pressure and dipsogenic effects of Ang II in the CNS.^5–7 Given the evidence that an increase in total Ca²⁺ current is involved in Ang II–stimulated neuronal activation,^8,9 we speculate that Ang II–induced O₂^•– production in central neurons is involved in Ang II–stimulated increases in [Ca²⁺]_i and the subsequent neuronal activation resulting in the physiological actions of Ang II in the brain. Recent studies by other groups lend support to this hypothesis. Sun et al showed that the O₂^•– scavenger Tempol or the NADPH oxidase inhibitor gp91ds-Tat markedly attenuated Ang II–mediated increases in neuronal firing rate of hypothalamic neurons.²⁵ In addition, Wang et al recently demonstrated that the Ang II–induced potentiation of L-type Ca²⁺ currents in neurons isolated from the nucleus of the solitary tract was blocked by O₂^•– scavenger MnTBAP, as well as by NADPH oxidase inhibitors gp91ds-Tat and apocynin.²⁶ These exciting new studies taken together with our current work strongly support the notion that NADPH oxidase-derived O₂^•– radicals are key signaling intermediates in the Ang II–induced increase in [Ca²⁺]_i and neuronal activation.

Although our present study demonstrates that Ang II–induced influx of extracellular Ca²⁺ in neurons involves O₂^•– production, we can only speculate about the mechanism by which this occurs. It is possible that O₂^•– itself acts on specific redox-sensitive amino acids of Ca²⁺ channels, thus altering either the opening or closing kinetics of the channels. ROS have been shown to increase neuronal Ca²⁺ current by regulating the opening of Ca²⁺ channels, possibly by oxidizing amino acid residues within the Ca²⁺ channel complex.¹¹ Moreover, Ciorba et al reported that the reversible oxidation of a methionine residue in a voltage-sensitive potassium channel modulated channel activity.¹³ An alternative hypothesis is that O₂^•– and other ROS alter membrane phospholipids, thereby modifying the ionic conductance of channel proteins.^27,28 Further studies will be required to determine how ROS, particularly O₂^•–, regulates Ang II–induced influx of extracellular Ca²⁺ and neuronal activation.

In addition to the notion that ROS induce increases in [Ca²⁺]_i in neurons, the reverse signaling mechanism has also been postulated, that is, ROS-induced Ca²⁺ leading to an increase in ROS in a feed-forward signaling loop. For example, Oyama et al demonstrated that H₂O₂ causes a dose-dependent increase in [Ca²⁺]_i in cerebellar neurons and that the Ca²⁺ ionophore ionomycin stimulated the production of ROS as measured by an increase in 2',7'-dichlorofluorescin fluorescence.²¹ Our DHE results demonstrating that Ang II stimulates an increase in O₂^•– production independent of Ang II–induced increases in [Ca²⁺]_i suggest this potential feed-forward loop does not operate in the context of Ang II responses in neurons.

Perspectives
This study provides direct evidence that Ang II–induced increases in [Ca²⁺]_i in neural cells are dependent on an increase in intracellular O₂^•– levels. Importantly, this increase in O₂^•– formation occurs independently of changes in cytosolic Ca²⁺ levels, suggesting that the radicals modulate Ca²⁺ entry. Although Neuro-2A cells provide an excellent model to study the intracellular signaling mechanisms of Ang II, O₂^•–, and Ca²⁺ in neurons, future studies in primary neurons are needed to confirm and extend the results of the present study to help in translating these findings to CNS neurons in vivo. Studies are ongoing in our laboratory and others to determine the functional significance of this interplay between Ang II, ROS, and Ca²⁺ in neurons. Furthermore, experiments designed to explore the target(s) of superoxide in mediating the Ang II–induced increase in [Ca²⁺]_i in central neurons will be of particular interest. Nevertheless, O₂^•– production in the central nervous system may be an important new therapeutic target in cardiovascular diseases associated with increased central Ang II signaling and neuronal activation, including hypertension and heart failure.

	Acknowledgments

 
This study was funded by grants from the NIH (HL-63887, HL-55006, and HL-14388 to R.L.D.) and American Heart Association (0540114N to R.L.D. and 0310039Z to M.C.Z.). The authors thank Dr Ramesh Bhalla for providing the facilities needed to perform the Ca²⁺ imaging experiments and for invaluable discussions.

Received October 7, 2004; first decision October 28, 2004; accepted November 16, 2004.

	References

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