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(Hypertension. 2005;45:276.)
© 2005 American Heart Association, Inc.

Scientific Contributions

Mechanisms of Angiotensin II–Mediated Decreases in Intraneuronal Ca²⁺ in Calcium-Loaded Stellate Ganglion Neurons

Stanley F. Fernandez; Ming-He Huang; Bruce A. Davidson; Paul R Knight, III; Joseph L. Izzo, Jr

From the Departments of Pharmacology (S.F.F., J.L.I.), Medicine (S.F.F., J.L.I.) and Anesthesiology (B.A.D., P.R.K.), School of Medicine and Biomedical Sciences, State University of New York at Buffalo; Department of Internal Medicine, Division of Cardiology (M.-H.H.), University of Texas Medical Branch, Galveston.

Correspondence to Joseph L. Izzo, Jr, MD, Department of Medicine, Erie County Medical Center, 462 Grider St, Buffalo, NY 14214. E-mail JIzzo{at}ams.ecmc.edu

	Abstract

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Our laboratory has reported previously that angiotensin II, type-1 (AT₁) receptor stimulation in isolated stellate ganglion neurons decreases intraneuronal calcium concentration ([Ca²⁺]i) acutely if baseline [Ca²⁺]i is high and increases [Ca²⁺]i if baseline [Ca²⁺]i is low. Part of the angiotensin II (Ang II) effect in high Ca²⁺ neurons is mediated through stimulation of Na⁺–Ca²⁺ exchange. Current experiments were conducted to identify additional steps in the signaling pathways. In Ca²⁺-loaded neurons, Ang II–induced decreases in [Ca²⁺]i were attenuated by phospholipase C inhibition (U73122) or nitric oxide (NO) synthase inhibition (L-NMMA) and were mimicked by the cGMP analogue 8-Br-cGMP. Protein kinase C (PKC) inhibition (bisindolylmaleimide I or Go6976) and protein kinase G (PKG) inhibition (KT5823) partially blocked Ang II–mediated decreases in [Ca²⁺]i, but complete blockade of Ang II effects was obtained with combined PKC and PKG inhibition. Modulation of inositol triphosphate (IP₃)-inducible ER Ca²⁺ release by [Ca²⁺]i was investigated using furaptra, an ER-retaining dye. IP₃-mediated ER Ca²⁺ release in ß-escin–permeabilized neurons was measured after clamping of [Ca²⁺]i from 50 nM to 800 nM. Maximal ER Ca²⁺ release was observed at 200 nM [Ca²⁺]i, with noted blunting of release at higher [Ca²⁺]i. Steady-state mRNA transcript and protein levels revealed that the principal IP₃R isoform expressed was IP₃R-II. These results suggest that Ca²⁺ loading in stellate ganglion neurons promotes Ang II-mediated decreases in [Ca²⁺]i via PKC and NO/cGMP/PKG pathways and inhibits IP₃R-II–mediated ER Ca²⁺ release.

Key Words: angiotensin II • calcium • signal transduction

	Introduction

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The sympathetic nervous system and the renin-angiotensin system are intricately interconnected pathways necessary for maintaining optimal cardiovascular and hemodynamic control. We have investigated the role of short-term angiotensin II (Ang II) treatment in rat stellate ganglion neurons and have found 2 opposing responses to angiotensin II, type-1 (AT₁) receptor activation that are dependent on baseline cytosolic calcium concentration ([Ca²⁺]i): increased [Ca²⁺]i in neurons with low baseline [Ca²⁺]i (stimulatory pattern) and decreased [Ca²⁺]i in neurons with high baseline [Ca²⁺]i (inhibitory pattern).¹ The latter response has been shown to require enhanced Na⁺–Ca²⁺ exchange (NCX),¹ but the pathways connecting AT₁ receptors to the membrane ion exchangers have not been identified.

The objective of our current study was to characterize intracellular signals involved in mediating Ang II–induced inhibitory responses using the ionomycin Ca²⁺-loaded model. AT₁ receptor stimulation in sympathetic neurons (like other tissues) generates inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol.² To characterize the potential role of IP₃/diacylglycerol-dependent pathways in Ang II–mediated decreases in [Ca²⁺]i, we inhibited several downstream elements, including protein kinase C (PKC), nitric oxide synthase (NOS), and protein kinase G (PKG). To characterize the role of Ca²⁺ loading on neuronal response to IP₃, permeabilized neurons were clamped at varying [Ca²⁺]i and IP₃-induced ER Ca²⁺ release was measured. To characterize the type of IP₃ receptor (IP₃R) expressed in neurons, IP₃R isoform-specific mRNA and protein quantitation were used.

	Materials and Methods

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Culture of Rat Stellate Ganglion Neurons
Details of this method have been described elsewhere.¹ Briefly, newborn (2- to 4-day old) Sprague–Dawley rats (Harlan) were anesthetized using 2% halothane. Stellate ganglion neurons were dissociated and resuspended in DMEM, F12, nerve growth factor-ß (100 ng/mL), penicillin/streptomycin, and 5% fetal bovine serum and plated onto laminin-coated coverslips. To enrich the number of neuronal cells, cultures were treated with cytosine arabinoside (20 µmol/L). Experiments were performed within 7 to 10 days of dissection. All appropriate Institutional Animal Care and Use Committee approvals were obtained before experimentation.

Measurement of [Ca²⁺]i
Global [Ca²⁺]i were estimated using Fura-2–based fluorescence imaging as previously described.³ Neurons were loaded with Fura-2-AM (Molecular Probes) and mounted onto an inverted microscope equipped with a cell chamber, kept at 37°C (FCS2; Bioptechs). A ratio-based microscopic fluorescence spectrophotometer (PTI) was used to excite the dye at 340/380 nm alternating at 2 Hz. Measurements of 510-nm emissions were recorded as fluorescence ratios (R_340/380) from a video screen encompassing a single neuronal soma. [Ca²⁺]i was calculated using the Grynkiewicz equation.⁴

Measurement of IP₃-Mediated ER Ca²⁺ Release
Assessment of IP₃-mediated Ca²⁺ release was based on a method previously described.⁵ In brief, the protocol involved loading cells with furaptra-AM, a Ca²⁺-sensitive endoplasmic reticulum (ER)-retaining fluorescent dye; cell membrane permeabilization using ß-escin; and IP₃ stimulation to induce ER Ca²⁺ release. Neurons were loaded with Furaptra-AM (45-minute; Molecular Probes), which accumulated in the cytoplasm and ER. Neurons were permeabilized using 40 µmol/L ß-escin (2-minute; Sigma). This allowed loss of cytoplasmic dye while retaining ER-localized dye and allowed introduction of IP₃ that is normally noncell-permeable. Neurons were stimulated with 10 µmol/L IP₃ at different experimental conditions. IP₃-mediated ER Ca²⁺ release was tracked by monitoring changes in fluorescence intensities with loss of fluorescence indicative of ER Ca²⁺ release. Fluorescence was measured using the same excitation/emission wavelength specifications for FURA 2 with data points taken at 2 Hz. The Ca²⁺ release rates were estimated based on the falling intensities fitted to a single exponential equation (SigmaPlot): ER Ca²⁺ release=Ae^–kt, with A being the amplitude normalized to 1, k the rate constant for fractional Ca²⁺ release, and t representing the time in seconds. The rate constant k (s^–1) was used to compare treatment groups. To account for nonspecific Ca²⁺ leak, control experiments were performed in the absence of IP₃. Resultant rates were based on the difference between experimental and control groups.

RNA Extraction and Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction
Total RNA, obtained by an acid guanidinium thiocyanate-phenol chloroform extraction method,⁶ was reverse-transcribed and amplified by real-time polymerase chain reaction (ABI Prism 5700 Sequence detection system; Applied Biosystems).⁷ Polymerase chain reaction primers were as follows: IP₃R-I (236 bp), 5'-CG-TTTCTCCTGCTGAGGTTC-3' and 5'-GTTCCCTCATCA-GCTTCTGC-3'; IP₃R-II (173 bp), 5'-ATTTGGACAGCCAGGT-CAAC-3' and 5'-GGCCACGACATCCTGTAACT-3'; and IP₃R-III, (247 bp) 5'-CATCAACGAGGACAATGTGG-3' and 5'-GCTGTCATGTCGACTCTCCA-3'. Threshold cycle numbers were obtained, representing the cycle number at which exponential growth of polymerase chain reaction products begin to be detected. Data were expressed relative to ß-actin, serving as endogenous mRNA control.

Immunocytochemistry
Neurons were fixed using 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with Super Block blocking buffer (1-hour; Pierce). Neurons were incubated with primary rabbit antibodies (24-hour; 1:300 dilution) against IP₃R-I, II, III isoforms and probed with fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies (6-hour). Images were captured by Sensys digital camera and fluorescence intensities estimated using Scion image software. Antibodies were obtained from Santa Cruz Laboratories.

Western Blot
Total protein was determined by Pierce bicinchoninic acid protein assay. Equal amounts of protein were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel. Proteins were electroblotted onto nitrocellulose membranes and blocked with 5% milk (1 hour). Membranes were incubated with primary rabbit antibodies against IP₃R-I,-II,-III (1-hour; 1:300 dilution) and probed with goat anti-rabbit horseradish peroxidase (1-hour, 1:5000 dilution), and bands were identified by enzyme-linked chemiluminescence reaction.

Data Analysis
Responses were expressed as means±SD. Two-tailed paired Student t test, ANOVA, and Tukey post-hoc test were used for statistical analyses, with P<0.05 (*) considered significant.

	Results

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Ang II–Induced Decreases in [Ca²⁺]i in Ca²⁺-Loaded Stellate Ganglion Neurons and Involvement of Phospolipase C
Stellate ganglion neurons were initially Ca²⁺-loaded using the Ca²⁺ ionophore, ionomycin. Neurons exposed to 1 µmol/L ionomycin (10-minute) in buffer solution containing 1.8 mmol/L Ca²⁺ raised mean baseline [Ca²⁺]i (nM) from 113±21 to 328±37. Ang II treatment (100 nM, 3-minute) resulted in a rapid decrease in [Ca²⁺]i (inhibitory response), [Ca²⁺]i (nM)=–126±47 (Figure 1A). Phospholipase C (PLC) involvement in Ang II–induced inhibitory responses was investigated using U73122, a competitive PLC inhibitor.⁸ Ca²⁺-loaded neurons were incubated with 10 µmol/L U73122 (30-minute), with resulting baseline [Ca²⁺]i (nM) of 328±37 and 307±16 for control and 10 µmol/L U73122, respectively. After U73122 pretreatment, Ang II-induced [Ca²⁺]i responses expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM) 38% (–126±47) and 11% (–35±12) for control and 10 µmol/L U73122. This corresponded to inhibition of Ang II responses by 71% (P=0.04; Figure 1B).

View larger version (11K): [in this window] [in a new window]   Figure 1. Involvement of PLC in Ang II–induced decrease in [Ca2+]i (inhibitory responses). A, Representative Ca2+ tracing of Ang II responses in neurons Ca2+-loaded using ionomycin. Ang II promoted decrease in [Ca2+]i that can be inhibited by 10 µmol/L U73122. Responses were re-established after removal of U73122 from perfusion buffer (data points at 2 Hz). B, Summary of U73122 effect on Ang II–induced inhibitory responses (n=7).

Role of Protein Kinase C in Ang II–Induced Inhibitory Responses
One of the downstream effector of PLC is PKC. The role of PKC in Ang II–mediated responses was investigated using bisindolylmaleimide-I (BIM), a competitive, nonisoform-specific inhibitor of PKC.⁹ Ca²⁺-loaded neurons were incubated with 10 µmol/L and 100 µmol/L BIM (30-minute), with resulting baseline [Ca²⁺]i (nM) of 320±4 and 312±2. After BIM pretreatment, Ang II–induced [Ca²⁺]i responses expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM) 46% (–152±26), 34% (–108±43), and 25% (–78±39) for control, 10 µmol/L and 100 µmol/L BIM, respectively. This corresponded to inhibition of Ang II responses by 26% (10 µmol/L BIM; P=0.07) and 46% (100 µmol/L BIM; P=0.02; Figure 2A). At micromolar concentrations, BIM has been reported to exhibit cross-inhibition of protein kinase A (PKA). Rp-cAMPS-TEA (100 µmol/L), a cell-permeable PKA inhibitor, did not change responses to 100 nM Ang II (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 2. Involvement of PKC in Ang II–induced inhibitory responses. Graph showing modulation of Ang II–induced inhibitory responses through inactivation of PKC by bisindolylmaleimide (BIM) (n=8) (A) or Go6976 (n=3) (B).

Involvement of Ca²⁺-sensitive isoforms of PKC (PKC-, -ß, -) was investigated using Go6976, an inhibitor of Ca²⁺-sensitive PKC isoforms.¹⁰ Ca²⁺-loaded neurons were incubated with 1 µmol/L and 10 µmol/L Go6976 (30-minute), with resulting baseline [Ca²⁺]i of (nM) 311±18 and 334±32. After Go6976 pretreatment, Ang II-induced [Ca²⁺]i responses expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM): 38% (–123±19), 23% (–73±22), and 10% (–34±16) for control, 1 µmol/L and 10 µmol/L Go6976, respectively. This corresponded to inhibition of Ang II responses by 39% (1 µmol/L Go6976; P=0.05) and 74% (10 µmol/L Go6976; P<0.01; Figure 2B).

Involvement of NO Pathway in Ang II–Induced Inhibitory Responses
Activation of the NO pathway has also been implicated in the reduction of [Ca²⁺]i in excitable cells.¹¹ Neurons were incubated with N^G-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that blocks nonisoform-specific NOS activity.¹² Ca²⁺-loaded neurons were incubated with 1 µmol/L, 10 µmol/L, and 100 µmol/L L-NMMA (30-minute), with resulting baseline [Ca²⁺]i (nM) of 356±45, 335±12, and 347±19. After L-NMMA pretreatment, Ang II-induced [Ca²⁺]i responses expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM) 40% (–130±28), 13% (–45±8), 7% (–25±4), and 3% (–11±4) for control, 1 µmol/L, 10 µmol/L, and 100 µmol/L L-NMMA, respectively. This corresponded to inhibition of Ang II responses by 68%, 83%, and 93% for 1 µmol/L, 10 µmol/L, and 100 µmol/L L-NMMA, respectively (P<0.01; Figure 3A).

View larger version (9K): [in this window] [in a new window]   Figure 3. NO/cGMP modulation of neuronal [Ca2+]i. A, Dose-dependent blockade of Ang II–induced inhibitory responses by L-NMMA (n=3/L-NMMA concentration). B, Graph demonstrating modulation of Ang II–induced inhibitory responses by KT5823 and combined KT5823/Go6976 treatment (n=5).

NO bioactivity is predominantly transduced by cGMP and PKG.¹¹ Involvement of PKG was assessed using KT5823, a competitive PKG inhibitor.¹³ Ca²⁺-loaded neurons were incubated with 1 µmol/L and 10 µmol/L KT5823 (30-minute), with resulting baseline [Ca²⁺]i (nM) of 311±24 and 308±13. After KT5823 pretreatment, Ang II-induced [Ca²⁺]i responses expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM) 38% (–123±19), 23% (–73±22), and 10% (–34±16) for control, 1 µmol/L and 10 µmol/L KT5823, respectively. This corresponded to inhibition of Ang II responses by 48% (1 µmol/L KT5823; P=0.04) and 72% (10 µmol/L KT5823; P=0.01; Figure 3B). Combined treatment with 10 µmol/L KT5823 and 10 µmol/L Go6976 inhibited Ang II responses by 96% (–5±8; P<0.01; Figure 3B).

Direct role of cGMP was examined by perfusing neurons with 8-Br-cGMP (10-minute), a stable, cell-permeable cGMP analogue.¹³ Ca²⁺-loaded neurons were incubated with 1 µmol/L, 10 µmol/L, and 100 µmol/L 8-Br-cGMP. [Ca²⁺]i responses to 8-Br-cGMP expressed as percent decrease in [Ca²⁺]i with respect to baseline were (, nM) 7% (–24±12; P=0.03), 34% (–110±21; P<0.01), and 50% (–162±18; P<0.01) for 1 µmol/L, 10 µmol/L, and 100 µmol/L 8-Br-cGMP, respectively. This concentration-dependent decrease in [Ca²⁺]i by 8-Br-cGMP mimics Ang II-induced inhibitory responses.

IP₃-Mediated ER Ca²⁺ Release Under Variable [Ca²⁺]i
A downstream effector of PLC is IP₃. IP₃ binds to ER-localized IP₃R to release intracellular Ca²⁺ stores into the cytoplasm. Modulation of IP₃R activation by high cytosolic Ca²⁺ was examined by quantifying IP₃-mediated ER Ca²⁺ release using furaptra-loaded neurons under variable [Ca²⁺]i (nM): 50, 80, 100, 200, 300, and 800 (Figure 4, see Methods section). A decrease in fluorescence intensity during IP₃ perfusion reflected the rate of Ca²⁺ loss from the ER. IP₃-mediated ER Ca²⁺ release under different [Ca²⁺]i conditions revealed differential release rates (x10^–2/s): 0.14±0.03 (50 nM), 0.27±0.1 (80 nM), 0.36±0.06 (100 nM), 1.24±0.3 (200 nM), 0.20±0.1 (300 nM), and 0.34±0.05 (800 nM). IP₃-mediated Ca²⁺ release, when plotted as a function of cytosolic Ca²⁺, exhibited a bell-shaped relationship with optimal rates occurring within the 200-nM range (Figure 4D). These findings demonstrate that cytosolic [Ca²⁺]i is an important modulator of IP₃-mediated ER Ca²⁺ release.

View larger version (31K): [in this window] [in a new window]   Figure 4. IP3-mediated ER Ca2+ release in single permeabilized stellate ganglion neurons. A, Fluorescence imaging of neurons used for IP3-mediated ER Ca2+ release. Neurons were loaded with Furaptra-AM dye, followed by membrane permeabilization with ß-escin. Furaptra-loaded neurons demonstrated homogeneous fluorescence intensities, suggesting adequate dye intake, whereas after ß-escin treatment, neurons showed granular perinuclear fluorescence consistent with ER localization. B, Typical Ca2+ tracing depicting key steps. Region enclosed by arrows represented neuronal response to IP3 under variable [Ca2+]i conditions. Rates were estimated using a single exponential equation (Ca2+ release=Ae–kt; see Methods section) representing IP3-induced ER Ca2+ release. C, Decline in fluorescence corresponded to fractional ER Ca2+ release. Neurons demonstrated differential responses to IP3 under different [Ca2+]i (50 to 800 nM, n=3 to 5/[Ca2+]i). D, Plotting of [Ca2+]i (x-axis) and IP3-induced ER Ca2+ release rates (y-axis) suggested peak rates at 200 nM [Ca2+]i.

Neuronal Expression of Isoform-Specific IP₃Rs
IP₃-mediated Ca²⁺ signaling pattern is dependent on the IP₃R isoform being expressed. mRNA levels of IP₃R isoforms (I, II, III) were quantified and expressed relative to ß-actin (see Methods section). mRNA concentrations for IP₃R-I,-II,-III were 4.5±0.1, 10.6±0.5, and 8.8±0.7, respectively (Figure 5A).

View larger version (28K): [in this window] [in a new window]   Figure 5. IP3R isoform expression profile. A, mRNA levels of IP3R isoforms were quantified using reverse-transcriptase polymerase chain reaction and expressed relative to ß-actin (n=3). B, Immunocytochemistry of IP3Rs (lower panel) double-labeled with filamentous-actin (F-actin, upper panel). C, Immunocytochemistry data expressed as IP3R fluorescence intensities relative to F-actin (n=10 to 15 cells/experiment from 3 independent experiments). D, Western blotting analysis of IP3R isoforms expressed as band intensities relative to moesin control. Insert showing bands corresponding to the different IP3R isoforms (n=3).

Protein levels were examined by immunocytochemistry using isoform-specific antibodies for IP₃R-I,-II,-III and relabeled with fluorescein isothiocyanate-conjugated secondary antibodies (Figure 5B). Fluorescence intensities of each isoform were expressed relative to filamentous actin. Relative intensities for IP₃R-I,-II,-III were 4.4±0.9, 11.9±4, and 8.9±1.0, respectively (Figure 5C).

Western blotting analyses of cell lysates for IP₃R isoforms revealed distinguishable bands near the 40-Kdal region, with the highest band intensity corresponding to IP₃R-II (Figure 5D). Each sample lane showed comparable sample loading using moesin as a loading control. IP₃R-I,-II,-III protein levels, relative to moesin, were 0.8±0.1, 1.1±0.1, and 0.9±0.1, respectively. Thus, primary rat stellate ganglion neurons as assessed by mRNA and protein levels exhibit differential expression of IP₃R-I,-II,-III, with IP₃R-II being the predominant form.

	Discussion

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In vivo, Ang II has been reported to either stimulate^14–16 or inhibit^17–19 the sympathetic nervous system (SNS). Our studies suggest that these opposing patterns are not artifactual but rather represent a complex response to Ang II that is governed by other metabolic and cellular influences. It is of note that the opposing whole-body response patterns seen with infused Ang II can be mimicked in single neurons in cell culture, which also respond to Ang II by either increasing or decreasing [Ca²⁺]i, depending on the baseline [Ca²⁺]i of the cell.¹ Previous work in our laboratory has demonstrated that AT₁ receptors are involved in this process, which eventuates in the stimulation of NCX in high-Ca²⁺ neurons. The current study identifies PKC and NO/cGMP/PKG pathways as intermediary mechanisms by which Ang II causes a decrease in [Ca²⁺]i when cells are Ca²⁺-loaded. Control of intraneuronal [Ca²⁺]i requires redundant mechanisms to maintain homeostatic levels. Ca²⁺ fluxes across plasma membranes and across intracellular storage compartments are highly responsive to the intracellular and extracellular environment. Differential activation of PKC/NO pathways under Ca²⁺-loaded condition provides additional mechanisms by which Ang II regulates global cytosolic Ca²⁺, a ubiquitous ion important for sympathetic neuronal function.

Specific downstream pathways by which AT₁ receptor-mediated signals are transduced include PLC-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate IP₃ and diacylglycerol. Each of these secondary signals sequentially activates downstream effectors that contribute to the ability of Ang II to influence [Ca²⁺]i. In the presence of low-baseline [Ca²⁺]i, IP₃ activates IP₃R to release intracellular Ca²⁺ stores, thus elevating [Ca²⁺]i and (presumably) activating neurotransmitter release.²⁰ In the presence of high-baseline [Ca²⁺]i, Ang II rapidly reduces [Ca²⁺]i via activation of PKC and NO pathways (Figures 2 and 3). Resting baseline [Ca²⁺]i, although highly variable, normally ranges from 100 nM to 300 nM. We have previously identified that when [Ca²⁺] is 200 nM, the stimulatory pattern is "switched" to the inhibitory pattern.¹ This response may reflect a homeostatic mechanism to "buffer" the effects of Ang II on individual neurons avoiding excessive activation and thereby stabilizing the SNS from marked variations.

Ang II–induced inhibitory responses occur primarily during high [Ca²⁺]i through activation of plasma membrane NCX.¹ Our current data demonstrating diminution of inhibitory responses after blockade of PKC and NO pathways suggest that these pathways may potentially act upstream of NCX or activate mechanisms independent of NCX. Both PKC^21–23 and NO^24–26 have been reported to independently activate NCX. A cross-talk between PKC and NO has also been suggested, in which PKC phosphorylates NOS, resulting in increased NOS activity.^27,28 Preferential activation of these 2 pathways under elevated cytosolic Ca²⁺ conditions (calcium sensing) may reside in the isoform-specific activation of PKC and NOS. PKCs are serine/threonine kinases that include several isoforms:²⁹ Ca²⁺-sensitive PKC (cPKC [, ß, and ]), non-Ca²⁺–sensitive PKC (nPKC [, , , and ]), and atypical PKC (aPKC [, /]). Figure 2 demonstrates that Ang II–induced inhibitory responses require the activation of a Ca²⁺-sensitive PKC, as evidenced by bisindolylmaleimide, and the specific Ca²⁺-sensitive PKC inhibitor, Go6976. These findings are consistent with previously reported immunohistochemical data demonstrating that sympathetic neurons predominantly express Ca²⁺-sensitive PKCs.³⁰ NOS, which catalyzes formation of NO, likewise exists as multiple isoforms:³¹ neuronal NOS (nNOS), inducible NOS, and endothelial NOS. Stellate ganglion neurons express mostly nNOS,³² an isoform that is highly Ca²⁺-sensitive.³¹ Elevated cytosolic Ca²⁺ conditions may therefore allow generation of Ang II–induced inhibitory responses through activation of Ca²⁺-sensitive mediators such as cPKC and nNOS.

Examination of IP₃R isoforms expressed in stellate ganglion neurons in this study reveals detectable levels of IP₃R-I, -II, and -III, but predominantly that of IP₃R-II (Figure 5). IP₃ activity depends principally on the predominant IP₃R isoform expressed, generating cell-type–specific Ca²⁺ signaling patterns. In general agreement with published literature,^5,33 IP₃R-II activation generates a characteristic Ca²⁺-sensitive release rates whereby at higher cytosolic Ca²⁺, IP₃-mediated responses are significantly blunted. This finding is consistent with the overall hypothesis that under elevated cytosolic Ca²⁺ conditions, Ang II will promote a net reduction in [Ca²⁺]i by the PKC/NO mechanisms and that the IP₃ pathway, which would tend to raise [Ca²⁺]i, would intuitively be blunted.

Limitations to the Present Study
Ionomycin-based Ca²⁺-loading technique provides a flexible means of establishing consistently elevated baseline [Ca²⁺]i amenable to signal transduction studies. Despite judicious titration to use only the lowest concentration of ionomycin (1 µmol/L) necessary, use of ionophores still poses real limitations. Ionomycin treatment results in acute Ca²⁺-loading that may not necessarily reflect conditions found in naturally persistent elevated Ca²⁺ conditions. Furaptra-based measurement of ER Ca²⁺ release involves permeabilizing neuronal cell membranes, which also results in loss of natural cytosolic environment including soluble proteins. Despite these limitations, this technique has been widely used, largely because of its flexibility for single-cell instantaneous recordings.³⁴ Alternative methodology such as use of isolated organelle preparations may allow direct quantitation of ER release but is severely limited by the multiple and harsh processing steps involved.

Perspectives
Existence of a "buffering mechanism" to protect the SNS against Ang II appears to exist at a cellular level. This property of isolated neurons appears to be a homeostatic mechanism that acts to stabilize SNS activity. Responses of isolated sympathetic neurons to Ang II are exquisitely sensitive to intraneuronal calcium content and a "switch mechanism" exists at 200 nM. Present findings may in part explain the well-known tachyphylaxis seen with prolonged Ang II infusion and the divergent SNS responses to Ang II that have been reported in human and animal experimentation.

Received November 5, 2004; first decision November 18, 2004; accepted December 2, 2004.

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