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编号:11168447
Adrenomedullin Stimulates Nitric Oxide Release from SK-N-SH Human Neuroblastoma Cells by Modulating Intracellular Calcium Mobilization
     Department of Cell Biology and Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

    Address all correspondence and requests for reprints to: Dr. Teresa L. Krukoff, Department of Cell Biology, Center for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: teresa.krukoff@ualberta.ca.

    Abstract

    We used SK-N-SH human neuroblastoma cells to test the hypothesis that adrenomedullin (ADM), a multifunctional neuropeptide, stimulates nitric oxide (NO) release by modulating intracellular free calcium concentration ([Ca2+]i) in neuron-like cells. We used a nitrite assay to demonstrate that ADM (10 pM to 100 nM) stimulated NO release from the cells, with a maximal response observed with 1 nM at 30 min. This response was blocked by 1 nM ADM22–52, an ADM receptor antagonist or 2 μM vinyl-L-NIO, a neuronal NO synthase inhibitor. In addition, 5 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular calcium chelator, eliminated the ADM-induced NO release. Similar results were observed when the cells were incubated in calcium-free medium or when L-type calcium channels were inhibited with 5 μM nifedipine or 10 μM nitrendipine. Depletion of calcium stores in the endoplasmic reticulum (ER) with 1 μM cyclopiazonic acid or 150 nM thapsigargin, or inhibition of ryanodine-sensitive receptors in the ER with 10 μM ryanodine attenuated the ADM-induced NO release. NO responses to ADM were mimicked by 1 mM dibutyryl cAMP, a cAMP analog, and were abrogated by 5 μM H-89, a protein kinase A inhibitor. Furthermore, Fluo-4 fluorescence-activated cell sorter analysis showed that ADM (1 nM) significantly increased [Ca2+]i at 30 min. This response was blocked by nifedipine (5 μM) or H-89 (5 μM) and was reduced by ryanodine (10 μM). These results suggest that ADM stimulates calcium influx through L-type calcium channels and ryanodine-sensitive calcium release from the ER, probably via cAMP-protein kinase A-dependent mechanisms. These elevations in [Ca2+]i cause activation of neuronal NO synthase and NO release.

    Introduction

    ADRENOMEDULLIN (ADM), a 52-amino-acid peptide, was originally described as a potent vasodilator (1), and a growing body of research has demonstrated that ADM, widely produced throughout the central nervous system (CNS) (2, 3), plays important roles in the maintenance of homeostasis through central mechanisms (4). For example, centrally administered ADM inhibits drinking and salt appetite (5, 6), suppresses food intake (7), and regulates blood pressure, heart rate, and baroreflex sensitivity by modulating autonomic activity (8, 9, 10, 11, 12, 13). Although it is becoming increasingly evident that central ADM modulates neuronal functions as a neurotransmitter, molecular mechanisms for ADM-induced effects in neurons and interactions between ADM and other neurotransmitters remain to be identified.

    Many of ADM’s actions in nonneural cells occur via ADM-stimulated synthesis of nitric oxide (NO). In vivo studies in rat (14), sheep (15), and humans (16) have suggested that ADM’s vasodilator effects are mediated in part by stimulation of NO release from endothelial cells. This idea has been confirmed by in vitro studies, which showed that ADM increases endothelial NO synthase (NOS) activity by elevating intracellular free calcium concentration ([Ca2+]i) (17, 18) or by activating phosphatidylinositol 3-kinase and protein kinase B/Akt (19). In cardiac myocytes, ADM augments NO production via a cAMP-dependent signaling pathway (20), and NO mediates an ADM-induced decrease in contractility of isolated myocytes (21). It has been shown that the antiapoptotic effect of ADM in human endothelial cells is abrogated by the NOS inhibitor, N-nitro-L-arginine methyl ester, and mimicked by the NO donor, sodium nitroprusside, suggesting that ADM inhibits programmed cell death through NO-mediated mechanisms (22). Finally, ADM has been shown to inhibit K+-stimulated aldosterone secretion from rat zona glomerulosa cells by stimulating endogenous NO synthesis (23).

    NO also acts as a nonconventional neurotransmitter in the CNS (24) and mediates some of the effects of central ADM. We have shown that hypotensive effects induced by microinjections of ADM into the paraventricular nucleus of the hypothalamus are attenuated by a neuronal NOS inhibitor or an endothelial NOS inhibitor (10), and that inhibition of NO synthesis by neuronal NOS eliminates ADM’s hypertensive effects in the rostral ventrolateral medulla (9). Although these findings suggest that ADM stimulates NO release from neurons, a direct demonstration of this ADM-NO cascade in neurons or neuron-like cells has not been made.

    In this study, neuron-like cells, SK-N-SH human neuroblastoma cells, were first shown to express the ADM receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP)-2 or -3 (25, 26). We then tested the hypothesis that ADM stimulates NO release from SK-N-SH cells by elevating calcium-dependent NOS activity. Furthermore, we investigated the mechanisms by which ADM modulates intracellular calcium dynamics to stimulate NO release. Finally, we determined whether ADM increases intracellular cAMP levels and whether the cAMP-protein kinase A (PKA) signaling pathway is required for ADM-induced effects on [Ca2+]i and NO release.

    Materials and Methods

    Materials

    SK-N-SH human neuroblastoma cells were obtained from American Type Culture Collection (Manassas, VA). All reagents for cell culture were purchased from Invitrogen Life Technologies, Inc. (Burlington, Canada). Fluo-4 AM and 2,3-diaminonaphthaline (DAN) were purchased from Molecular Probes (Eugene, OR). ADM, ADM22–52, and calcitonin gene-related peptide 8–37 were purchased from American Peptide (Sunnyvale, CA). Vinyl-L-NIO was obtained from Alexis (San Diego, CA), and L-NIO was purchased from Tocris Cookson (Ellisville, MO). 2-Aminoethoxydiphenylborate (2-APB) was obtained from Calbiochem (San Diego, CA). Nifedipine, nitrendipine, 1400W, cyclopiazonic acid (CPA), thapsigargin, ryanodine, dibutyryl cAMP (dBcAMP), and H-89 were purchased from Sigma-Aldrich Corp. (Oakville, Canada).

    Cell culture

    As we have described previously (27), SK-N-SH cells were cultured at 37 C in a humidified atmosphere of 5% CO2 and 95% O2 in Eagle’s MEM, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 g/ml streptomycin. Medium was changed every 2–3 d. Subconfluent cells were harvested and incubated in phenol red-free MEM supplemented with 5% charcoal/dextran-stripped fetal calf serum for 48 h, then used in the following experiments.

    Expression of ADM receptor components by RT-PCR

    To determine whether ADM receptors are expressed in SK-N-SH cells, mRNAs of ADM receptor components were detected in untreated cells using RT-PCR. Total RNA was extracted with a TriPure Isolation Reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions and reverse transcribed to cDNA, as described previously (28). Five microliters of cDNA were used in subsequent 50-μl PCRs. The PCR solution contained 2.5 U Taq DNA polymerase (Biocompare, Inc., South San Francisco, CA), 200 μM deoxy-NTPs, 2 mM MgCl2, and 250 nM of each primer. The sequences of primers, annealing temperatures, and product sizes are described in Table 1. After an initial 5-min incubation at 94 C, 40 cycles were run consisting of 45 sec at 94 C, 45 sec at an annealing temperature appropriate for each primer pair, and extension for 2 min at 72 C, followed by final extension at 72 C for 10 min. To rule out the possibility of amplifying genomic DNA, some RT-PCRs were carried out in the absence of the reverse transcriptase. Products were analyzed by standard electrophoresis.

    TABLE 1. PCR amplification of the ADM receptor components: sequences of oligonucleotide primers, annealing temperatures, and expected product sizes

    Evaluation of NO release with DAN fluorometric nitrite assay

    Cells, grown at a density of 105 cells/well in six-well culture plates, were washed twice with phenol red-free and serum-free MEM and subjected to various treatments (as described below). Using a modified DAN fluorometric nitrite assay, total NO release was estimated based on the levels of nitrite in the medium as we have previously described (27). Briefly, duplicate samples of medium (500 μl each) were collected from each well and mixed with 40 μl freshly prepared DAN (50 μg/ml in 0.62 M HCl), then incubated for 20 min at room temperature. The reaction was terminated with 50 μl 2.8 M NaOH. The intensity of fluorescence was measured using a spectrofluorometer (SLM-Aminco, model 8100, SLM Instruments, Inc., Rochester, NY) with excitation at 365 nm and emission at 450 nm. The nitrite levels in the samples were calculated on the basis of the standard curve prepared with sodium nitrite (27).

    Dose response and time course of ADM-induced NO release

    In dose-response experiments, cells were treated with 10 pM to 100 nM ADM or vehicle for 30 min, and nitrite levels in the medium were measured. Temporal changes in nitrite levels were examined 5, 10, 20, 30, or 60 min after treatment with 1 nM ADM.

    Identification of receptors required for ADM-induced NO release

    To determine which receptors are responsible for ADM’s effect on NO release, cells were treated with 1 nM ADM in the presence of 1 nM ADM22–52 (a putative ADM receptor antagonist), 1 nM CGRP8–37 (a putative CGRP receptor antagonist), or vehicle for 30 min, and nitrite levels were measured.

    Selective inhibition of NOS isoforms and ADM-induced NO release

    To determine which NOS isoform(s) is recruited by ADM to synthesize NO, cells were pretreated with the respective NOS inhibitors for 30 min, followed by treatment with 1 nM ADM in the presence of the same inhibitor for 30 min. Vinyl-L-NIO (2 μM) was used as a selective neuronal NOS inhibitor because the IC50 of vinyl-L-NIO for neuronal NOS is 0.1 μM, making it 120- and 600-fold more sensitive for neuronal NOS than for endothelial NOS and inducible NOS, respectively (29). L-NIO, used at 1 μM, is the only endothelial NOS inhibitor (IC50 = 0.5 μM) currently available, providing 8- and 4-fold more selectivity for endothelial NOS than for neuronal NOS and inducible NOS, respectively (30). Inducible NOS was inhibited by 1 or 5 μM 1400W (IC50 = 0.23 μM), which shows 31- and 4000-fold more sensitivity for inducible NOS than for endothelial NOS and neuronal NOS, respectively (31).

    Modulation of [Ca2+]i and ADM-induced NO release

    To investigate whether ADM-induced NO release is calcium dependent, cells were pretreated with 5 or 50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; an intracellular calcium chelator) for 30 min to chelate free calcium in the cytoplasm as we have described (27), followed by treatment with 1 nM ADM plus BAPTA-AM for 30 min; nitrite levels were measured thereafter. In some experiments, nitrite levels were assessed after cells were incubated in calcium-free medium containing 1 nM ADM for 30 min to evaluate the role of extracellular calcium in ADM-induced NO release. To demonstrate recruitment of L-type voltage-dependent calcium channels (VDCC) and intracellular calcium stores in the endoplasmic reticulum (ER) in ADM’s effects, cells were pretreated with specific pharmacological agents for 30 min, followed by treatment with 1 nM ADM plus the same agent for 30 min, and nitrite levels were measured. Nifedipine (5 μM) (27) or 10 μM nitrendipine (32) was used to inhibit the L-type VDCC. In experiments assessing the involvement of calcium in the ER, the ER-associated calcium-adenosine triphosphatase (calcium-ATPase) inhibitor, 1 μM CPA (33), or 150 nM thapsigargin (34) was applied to deplete calcium stores in the ER. Ryanodine [10 μM; a ryanodine-sensitive receptor (RyR) inhibitor] was used to determine the role of RyR (35), and the recruitment of inositol 1,4,5-trisphosphate receptor (IP3R) was determined using 10 or 100 μM 2-APB (IP3R inhibitor) (36).

    cAMP-PKA signaling pathway and ADM-induced NO release

    To compare the effects of ADM and cAMP on NO release, nitrite levels were measured after cells were treated with 1 nM ADM, 1 mM dBcAMP (a cell-permeable cAMP analog) (37), or vehicle for 30 min. To evaluate the role of PKA in ADM’s effects on NO release, cells were pretreated with 5 μM H-89, a PKA inhibitor, for 30 min (38), followed by treatment with 1 nM ADM plus 5 μM H-89 for 30 min, and nitrite levels were measured.

    Monitoring of intracellular calcium dynamics by Fluo-4 fluorescence-activated cell sorter (FACS) analysis

    To investigate the effects of ADM on [Ca2+]i, we monitored intracellular calcium dynamics in SK-N-SH cells using a modified Fluo-4 FACS analysis (39, 40, 41). Cells were trypsinized and suspended at 2.5 x 106 cells/ml in phenol red-free and serum-free MEM in flow cytometry tubes (500 μl/tube). After a 2-h recovery at 37 C, the cells were loaded with 5 μM Fluo-4 AM, a fluorescent calcium indicator, for 30 min at room temperature. The cells were preincubated in MEM-containing vehicle, 5 μM nifedipine, 10 μM ryanodine, or 5 μM H-89 for 30 min at 37 C, and basal levels of fluorescence intensity (excitation at 488 nm and emission at 525 nm) were recorded with a flow cytometer (FACScan, BD Biosciences, Franklin Lakes, NJ). ADM was then added to the medium at a final concentration of 1 nM, and the intensity of fluorescence was recorded every 5 min for 30 min thereafter.

    Measurement of intracellular cAMP by enzyme immunoassay

    Cells, grown at a density of 105/well in 96-well culture plates, were treated with 0.5 mM 3-isobutyl-1-methylxanthine (a cyclic nucleotide phosphodiesterase inhibitor; Sigma-Aldrich Corp.) for 30 min to prevent breakdown of accumulated cAMP, followed by treatment with 1 nM ADM or vehicle for 10, 20, or 30 min. Intracellular cAMP levels were measured with a commercial enzyme immunoassay kit according to the manufacturer’s instructions (Amersham Biosciences, Arlington Heights, IL).

    Statistical analysis

    Data are presented as the mean ± SEM. Statistical analyses were carried out with SigmaStat software (Jandel Corp., San Ramon, CA). Depending on the experiments, differences among groups were determined by one- or two-way ANOVA, followed by post hoc Tukey’s test when the ANOVA indicated significant differences. P < 0.05 indicated statistical significance.

    Results

    SK-N-SH cells express mRNAs of ADM receptor components

    Figure 1 shows that mRNAs of all the known ADM receptor components, CRLR, RAMP-2, and RAMP-3, were detected by RT-PCR in untreated SK-N-SH cells. In the reactions carried out in the absence of reverse transcriptase, no cDNA product was generated (data not shown), demonstrating that the RNA was not contaminated by genomic DNA. These results demonstrate that SK-N-SH cells express ADM receptors and suggest that these cells are able to respond to ADM.

    FIG. 1. Expression of ADM receptor components in SK-N-SH cells. An ethidium bromide-stained 1% agarose gel shows cDNA amplified from RNA of SK-N-SH cells with human CRLR-, RAMP-2-, and RAMP-3-specific primers.

    ADM stimulates NO release from SK-N-SH cells

    Treatment of cells with ADM (10 pM to 100 nM) for 30 min significantly increased nitrite levels in the medium compared with control levels. In particular, ADM at 10 pM, 100 pM, and 1 nM dose-dependently increased nitrite levels, with the maximal effect observed at 1 nM; the responses to ADM at 10 and 100 nM were comparable to the response to 1 nM ADM (Fig. 2A). In the time-course experiments, ADM (1 nM) significantly increased nitrite levels at 20, 30, and 60 min compared with the respective control values, with the most robust increase occurring at 30 min; treatment with ADM for 5 and 10 min did not evoke significant increases in nitrite levels (Fig. 2B). Based on these results, treatment with ADM (1 nM) for 30 min was used in subsequent experiments.

    FIG. 2. A, Dose response of ADM-induced NO release from SK-N-SH cells. Cells were treated with vehicle (control) or ADM at the indicated concentrations. Nitrite levels in the culture medium were measured 30 min after treatment. B, Time course of ADM-induced NO release. Nitrite levels in the culture medium were measured at the indicated times after treatment of cells with vehicle (control) or ADM (1 nM). C, Effects of ADM22–52 and CGRP8–37 on ADM-induced NO release. Nitrite levels in the culture medium were measured 30 min after treatment of cells with vehicle (controls) or ADM (1 nM) in the presence of vehicle, ADM22–52 (1 nM), or CGRP8–37 (1 nM), respectively. n = 6 in each group. *, P < 0.05; **, P < 0.01 (vs. the respective control).

    ADM-induced NO release is abolished by ADM22–52, but not by CGRP8–37

    The potential role of ADM receptors and CGRP receptors in ADM-induced NO release was evaluated using the ADM receptor antagonist, ADM22–52, and the CGRP receptor antagonist, CGRP8–37. The ADM-induced increase in nitrite levels was completely inhibited by cotreatment with ADM22–52, whereas CGRP8–37 had no effect (Fig. 2C), indicating that ADM-induced NO release is mediated by ADM receptors, but not by CGRP receptors. Treatment with ADM22–52 or CGRP8–37 alone did not change basal nitrite levels compared with the control values (Fig. 2C).

    Inhibition of neuronal NOS abolishes ADM-induced NO release

    Recruitment of NOS isoforms in ADM-induced NO release was investigated using selective NOS inhibitors. As shown in Fig. 3A, inhibition of neuronal NOS with 2 μM vinyl-L-NIO abolished the ADM-induced increase in nitrite levels. In contrast, neither L-NIO (1 μM; the endothelial NOS inhibitor) nor 1400W (1 or 5 μM; the inducible NOS inhibitor) affected the ADM-induced effect (Fig. 3, B and C). Together these results indicate that activation of neuronal NOS contributes to ADM-induced NO release, whereas endothelial NOS and inducible NOS are not involved. Note that basal levels of nitrite levels were not changed by the NOS inhibitors alone (Fig. 3).

    FIG. 3. Effects of selective NOS inhibitors on ADM-induced NO release from SK-N-SH cells. Cells were pretreated with vehicle, 2 μM vinyl-L-NIO (A), 1 μM L-NIO (B), or 1 or 5 μM 1400W (C) for 30 min, followed by 30-min treatment with vehicle (control) or ADM (1 nM) in the presence of vehicle, 2 μM vinyl-L-NIO (A), 1 μM L-NIO (B), or 1 or 5 μM 1400W (C), respectively. Nitrite levels in the medium were measured. n = 4–6 in each group. *, P < 0.05; **, P < 0.01 (vs. the respective control).

    Chelation of free calcium in cytoplasm abolishes ADM-induced NO release

    BAPTA-AM, an intracellular calcium chelator, was used to demonstrate the importance of intracellular calcium in ADM-induced NO release. Figure 4 shows that BAPTA-AM (5 or 50 μM) completely inhibited the increase in nitrite levels evoked by ADM, indicating that ADM-induced NO release is a calcium-dependent event. Consistent with our previous observation (27), BAPTA-AM itself significantly increased basal nitrite levels compared with vehicle (Fig. 4).

    FIG. 4. Effects of BAPTA-AM on ADM-induced NO release from SK-N-SH cells. SK-N-SH cells were pretreated with vehicle or BAPTA-AM (5 or 50 μM) for 30 min, followed by 30-min treatment with vehicle (control) or ADM (1 nM) in the presence of vehicle or BAPTA-AM (5 or 50 μM), respectively. Nitrite levels in the medium were measured. n = 4 in each group. **, P < 0.01 (vs. the respective control).

    Removal of extracellular calcium or blockade of L-type VDCC abolishes ADM-induced NO release

    The involvement of extracellular calcium in ADM-induced NO release was investigated using calcium-free medium or with application of nifedipine or nitrendipine to block the L-type VDCC. When extracellular calcium was removed by incubation of cells in calcium-free medium, basal nitrite levels were reduced compared with those in cells incubated in calcium-containing medium, and the ADM-induced increase in nitrite levels was completely inhibited (Fig. 5A). A similar inhibition was observed in experiments using 5 μM nifedipine or 10 μM nitrendipine, whereas nifedipine or nitrendipine alone did not change basal levels of nitrite in the medium (Fig. 5, B and C). These results indicate that calcium influx from the L-type VDCC is required for ADM-induced NO release.

    FIG. 5. Effects of calcium-free medium, nifedipine, and nitrendipine on ADM-induced NO release from SK-N-SH cells. A, Cells incubated in calcium-containing or calcium-free medium were treated with vehicle (control) or ADM (1 nM) for 30 min. B and C, Cells were pretreated with vehicle, 5 μM nifedipine (B), or 10 μM nitrendipine (C) for 30 min, followed by 30-min treatment with vehicle (control) or ADM (1 nM) in the presence of vehicle, 5 μM nifedipine (B), or 10 μM nitrendipine (C), respectively. Nitrite levels in the medium were measured. n = 4 in each group. **, P < 0.01 (vs. the respective control).

    Depletion of calcium stores in ER or inhibition of RyR attenuates ADM-induced NO release

    ER-associated calcium-ATPase inhibitors (CPA and thapsigargin), a RyR inhibitor (ryanodine), and an IP3R inhibitor (2-APB) were used to evaluate the role of calcium stores in the ER in the ADM-induced NO release. The ADM-induced increase in nitrite levels was significantly attenuated, but not completely abolished, by pretreatment with 1 μM CPA or 150 nM thapsigargin (Fig. 6, A and B). A similar reduction in the ADM-induced response was observed when cells were pretreated with 10 μM ryanodine (Fig. 6C). Pretreatment with 10 and 100 μM 2-APB did not affect ADM’s effect on NO release, although 2-APB (100 μM) alone significantly decreased basal levels of nitrite in the medium (Fig. 6D). These results indicate that the ADM-induced NO release from SK-N-SH cells is mediated partly by calcium release from the ER via the RyR, but not via the IP3R.

    FIG. 6. Effects of CPA, thapsigargin, ryanodine, and 2-APB on ADM-induced NO release from SK-N-SH cells. Cells were pretreated with vehicle, 1 μM CPA (A), 150 nM thapsigargin (B), 10 μM ryanodine (C), or 10 or 100 μM 2-APB (D) for 30 min, followed by 30-min treatment with vehicle (control) or ADM (1 nM) in the presence of vehicle, 1 μM CPA (A), 150 nM thapsigargin (B), 10 μM ryanodine (C), or 10 or 100 μM 2-APB (D), respectively. Nitrite levels in the medium were measured. n = 4 in each group.*, P <0.05; **, P < 0.01 (vs. the respective control). #, P < 0.05; ##, P < 0.01 (vs. vehicle-preincubated cells).

    ADM-induced NO release is mimicked by a cAMP analog and is abolished by inhibition of PKA activity

    Treatment with 1 mM dBcAMP, a cAMP analog, for 30 min significantly increased nitrite levels in the medium; this response was comparable to the effect of 1 nM ADM (Fig. 7B). Furthermore, the ADM-induced increase in nitrite levels was abolished by inhibition of PKA with 5 μM H-89, whereas H-89 itself did not influence basal nitrite levels (Fig. 7C). Taken together, these data indicate that the cAMP-PKA signaling pathway is required for ADM-induced NO release.

    FIG. 7. A, Effects of ADM on intracellular cAMP levels in SK-N-SH cells. Cells were treated with 0.5 mM 3-isobutyl-1-methylxanthine for 30 min, followed by treatment with vehicle (control) or 1 nM ADM for the indicated times. Intracellular cAMP levels were measured with a commercial enzyme immunoassay kit. B, Effects of dBcAMP on NO release from SK-N-SH cells. Cells were treated with vehicle, 1 nM ADM, or 1 mM dBcAMP for 30 min. C, Effects of H-89 on ADM-induced NO release from SK-N-SH cells. Cells were pretreated with vehicle or 5 μM H-89 for 30 min, followed by 30-min treatment with vehicle (control) or 1 nM ADM in the presence of vehicle or 5 μM H-89. Nitrite levels in the medium were measured. n = 4–6 in each group. *, P <0.05; **, P < 0.01 (vs. the respective control).

    ADM modulates intracellular calcium mobilization in SK-N-SH cells

    Using Fluo-4 FACS analysis, we showed that treatment of cells with 1 nM ADM for 30 min caused a significant increase in [Ca2+]i (Fig. 8, A and B). Although the effects of ADM before 30 min were not significantly different from the control values, trends toward increased [Ca2+]i were observed at 10, 15, 20, and 25 min after treatment (data not shown).

    FIG. 8. Effects of ADM on intracellular calcium mobilization in SK-N-SH cells using Fluo-4 FACS analysis. Cells were loaded with 5 μM Fluo-4 AM, then pretreated with vehicle (A and B), 5 μM nifedipine (C), 10 μM ryanodine (D), or 5 μM H-89 (E) for 30 min, and basal levels of fluorescence intensity were read in a flow cytometer (). Vehicle (A) or 1 nM ADM (B–E) was then applied to the cells, and fluorescence intensities were read again 30 min later (). F, Statistical analysis of data from four independent experiments. *, P < 0.05 (vs. the respective basal level). #, P < 0.05 (vs. vehicle-pretreated cells).

    The roles of the L-type VDCC, RyR, and PKA in ADM-induced intracellular calcium accumulation were demonstrated using respective inhibitors. The ADM-induced increase in [Ca2+]i was abolished with 5 μM nifedipine (Fig. 8C) and 5 μM H-89 (Fig. 8E), whereas 10 μM ryanodine attenuated this effect (Fig. 8D). These results suggest that both the L-type VDCC and PKA are required for ADM-induced intracellular calcium accumulation, and that RyR is also involved in this response. Note that pretreatment with nifedipine or ryanodine alone did not affect basal [Ca2+]i (Fig. 8, C and D). Consistent with observations in muscle cells (42) and lymphocytes (43), H-89 itself significantly increased basal levels of [Ca2+]i in SK-N-SH cells (Fig. 8E).

    ADM elevates intracellular cAMP levels

    Temporal responses of intracellular cAMP levels to 1 nM ADM were investigated at 10, 20, and 30 min. Treatment with ADM for 20 min significantly increased intracellular cAMP levels. Effects of ADM at 10 and 30 min were not significantly different from the control values, although trends toward elevated cAMP levels were observed (Fig. 7).

    Discussion

    In the present study we demonstrate for the first time that ADM stimulates NO release from neuron-like cells. By monitoring NO release and intracellular calcium dynamics evoked by ADM, we show that ADM stimulates NO release by recruiting both extracellular and intracellular calcium stores to increase [Ca2+]i and thus to activate neuronal NOS. Finally, we provide evidence that the cAMP-PKA signaling pathway mediates the effects of ADM on [Ca2+]i and NO.

    SK-N-SH cells are used as a model for investigating ADM-induced NO release from neurons

    We chose SK-N-SH human neuroblastoma cells as a model to investigate the effects of ADM on NO release from neurons for several reasons. First, we have shown that SK-N-SH cells express all of the known NOS isoforms (27). Second, these cells have been successfully used to address questions about NO release from neurons (27, 44, 45, 46, 47, 48). Third, we show with RT-PCR that mRNAs of ADM receptor components, CRLR, RAMP-2, and RAMP-3, are present in the cells, suggesting that SK-N-SH cells are capable of responding to ADM. This finding is consistent with previous observations that cells of another neuroblastoma cell line, SK-N-MC, express CRLR and RAMP-2 (49) and respond to ADM stimulation (50). Based on these results, we consider SK-N-SH cells to be an appropriate in vitro model to investigate the effects of ADM on NO release from neurons. Because SK-N-SH cells originate from tumor tissue of human neuroblastoma, however, we recognize that our results may not necessarily represent all responses of neurons.

    ADM stimulates NO release from SK-N-SH cells through ADM receptor-mediated mechanisms

    The results from our previous in vivo experiments first led us to hypothesize that ADM stimulates NO release from neurons. We showed that a proportion of neuronal NOS-positive neurons in the paraventricular nucleus also express ADM receptors (10), that intracerebroventricular (icv) injections of ADM activate NOS-positive neurons in the paraventricular nucleus (8), and that icv ADM stimulates NO production in the hypothalamus (8). Although all of these in vivo findings suggest that ADM stimulates NO release from neurons, direct evidence for this event was not available before the current study. By monitoring nitrite levels in the culture medium, a method that has been widely used to study NO release from cells (27, 51, 52, 53), we provide the first evidence that ADM indeed stimulates NO release from neuron-like cells. This ADM-induced NO release may underlie the cardiovascular effects of ADM in the paraventricular nucleus and rostral ventrolateral medulla, because ADM’s effects in these nuclei are attenuated or abolished by NOS inhibitors (9, 10). Furthermore, because NO at low concentrations has been shown to mediate protective effects of estrogen in SK-N-SH cells (54) and to rescue SK-SY5Y neuroblastoma cells from oxidative stress (55), the modest increase in NO release induced by ADM may also mediate the neuroprotective effects of ADM that have been observed in ischemic brain (56, 57).

    Although 10 pmol to 1 nM ADM stimulate NO release in a dose-dependent manner, ADM at doses higher than 1 nM does not induce greater responses. This response pattern probably occurs because ADM’s effects are mediated by receptor-dependent mechanisms, and 1 nM ADM is sufficient to occupy all binding sites for ADM on the cell surface. It is clear that ADM receptors mediate the NO response to ADM, because inhibition of ADM receptors with ADM22–52 eliminates ADM-induced NO release. Besides ADM receptors, ADM has been shown to have appreciable affinity for CGRP receptors (58), which are comprised of CRLR and RAMP-1 (25, 26). However, we show that CGRP8–37, the CGRP receptor antagonist, does not affect ADM-induced NO release, demonstrating that CGRP receptors are not involved.

    Consistent with previous studies using the same nitrite assay (27, 52), we found that control (vehicle-treated) cultures also show increased NO release in a time-dependent manner; the amount of NO released is comparable with that in previous reports. This NO release is probably due to basal NOS activities, which have been demonstrated in unstimulated SK-N-SH cells (27). Because removal of extracellular calcium or inhibition of IP3R-mediated calcium release from the ER decreases basal nitrite levels, calcium-sensitive NOS activities (neuronal and/or endothelial NOS) are probably involved in basal NO release.

    Neuronal NOS is required for ADM-induced NO release from SK-N-SH cells

    All of the known NOS isoforms have been demonstrated to contribute to ADM-induced NO production in various types of cells. It is well established that endothelial NOS in vascular endothelial cells is recruited by ADM to stimulate NO synthesis and thus to mediate vasodilatation (17, 18, 19). Experiments in cardiac myocytes demonstrated that the expression of inducible NOS and the production of NO are augmented by ADM (20). Although our recent in vivo studies suggested that neuronal NOS activity contributes to ADM-induced effects in the brain (8, 9, 10), the mechanisms of neuronal NOS recruitment by ADM in neurons or neuron-like cells had not been elucidated before the current study.

    Because all of the known NOS isoforms exist in SK-N-SH cells (27), the involvement of specific NOS isoforms in ADM-induced NO release was assessed using selective NOS inhibitors. Inhibition of neuronal NOS with vinyl-L-NIO completely blocks ADM’s effect on NO release, whereas this effect is not affected by either L-NIO (an endothelial NOS inhibitor) or 1400W (an inducible NOS inhibitor). Thus, we demonstrate for the first time that neuronal NOS is recruited by ADM to synthesize NO in neuroblastoma cells. Consistent with these results, we previously showed that icv injections of ADM activate neuronal NOS-positive neurons in the paraventricular nucleus and increase NO production in the hypothalamus (8), and that ADM’s effects in the paraventricular nucleus (10) and rostral ventrolateral medulla (9) are mediated at least in part by NO derived from neuronal NOS.

    ADM induces NO release by elevating [Ca2+]i

    Although ADM has been shown to increase [Ca2+]i in nonneural cells (17, 18, 59, 60), the effects of ADM on [Ca2+]i in neuron-like cells had not been described. Because it is well established that NO production by neuronal NOS is triggered by elevations in [Ca2+]i (61), the recruitment of neuronal NOS in ADM-induced NO release suggests that ADM should increase [Ca2+]i in neuroblastoma cells. Using Fluo-4 FACS analysis, we show that 1 nM ADM tends to increase [Ca2+]i 10 min after treatment, and that this increase becomes significantly different from control levels by 30 min. Considering that 1 nM ADM begins to stimulate NO release from 20 min and that the most robust increase occurs at 30 min, these results suggest that increases in [Ca2+]i and NO release evoked by ADM are correlated. More importantly, we show that the ADM-induced NO release is abolished when free calcium in the cytoplasm is chelated by BAPTA-AM. Taken together, these results provide evidence that ADM stimulates neuronal NOS activity by stimulating an increase in [Ca2+]i in SK-N-SH cells, as illustrated by the proposed model in Fig. 9.

    FIG. 9. Proposed mechanisms for ADM-induced NO release from SK-N-SH cells. Binding of ADM to its receptors leads to calcium influx through the L-type VDCC. Elevated cytoplasmic calcium then acts upon the RyR to trigger additional calcium release from the ER. Increases in [Ca2+]i facilitate activation of neuronal NOS, which, in turn, leads to synthesis and release of NO. The cAMP-PKA signaling pathway mediates the ADM-induced responses, possibly via its actions on the L-type VDCC, RyR, or ER-associated calcium-ATPase. ADM R, ADM receptor; nNOS, neuronal NOS.

    Although it has been established that activities of both neuronal and endothelial NOS are calcium dependent (61), our data show that only neuronal NOS is activated by the ADM-induced increase in [Ca2+]i. Similar to our results, neuronal NOS has been reported to be much more active than endothelial NOS in HEK 293 cells when [Ca2+]i is increased (62), indicating that even in the same cellular environment, activities of neuronal and endothelial NOS may be differentially regulated by intracellular calcium mobilization.

    Extra- and intracellular calcium stores are both recruited by ADM to stimulate NO release

    ADM increases [Ca2+]i in nonneural cells both by stimulating calcium influx from the extracellular medium and inducing calcium release from the ER (17, 18, 59), but it was not known which calcium sources are recruited by ADM to induce NO release in neuroblastoma cells. In the present study we show that calcium influx is crucial for ADM-induced NO release by demonstrating that removal of extracellular calcium eliminates the NO response. In regard to which calcium channel is responsible for this ADM-induced calcium influx, the role of the L-type VDCC, which is expressed in SK-N-SH cells (63), was evaluated. By showing that inhibition of the L-type VDCC with nifedipine or nitrendipine abolishes both the intracellular calcium accumulation and NO release evoked by ADM, we demonstrate that opening of the L-type VDCC mediates calcium influx and thus contributes to ADM-induced NO release. Consistent with our results, calcium influx via the L-type VDCC has been previously reported to be involved in ADM’s positive inotropic effect in an isolated rat heart preparation (59).

    Besides calcium influx through the cell membrane, we show that calcium release from the ER plays a role in the ADM-induced effects in neuroblastoma cells by demonstrating that the ADM-induced NO release is attenuated when the ER calcium stores are depleted with CPA or thapsigargin. We also show that the ADM-induced increases in [Ca2+]i and NO release are attenuated by ryanodine, demonstrating that calcium release stimulated by ADM occurs via RyR, the calcium-gated calcium release channel located on the ER (64, 65). A similar phenomenon has been observed in an isolated rat heart preparation, in which the inotropic effect of ADM is attenuated by either thapsigargin or ryanodine (59). We have ruled out the possibility that the ADM-induced NO release involves IP3R-mediated calcium release from the ER with our finding that inhibition of IP3R by 2-APB does not influence NO response to ADM treatment.

    It is worthwhile to emphasize that elimination of calcium influx, using calcium-free medium, nifedipine, or nitrendipine, abolishes ADM-induced responses in [Ca2+]i and subsequent NO release. Interestingly, inhibition of calcium release from the ER by CPA, thapsigargin, or ryanodine only reduces, but does not abolish, ADM’s effects. Therefore, our results indicate that although both extracellular and intracellular calcium sources contribute to ADM-induced effects, only extracellular calcium is indispensable. Thus, we propose that RyR-mediated calcium release from the ER is triggered by, and thus dependent on, ADM-induced calcium influx via the L-type VDCC (Fig. 9). In support of this interpretation, RyR has been shown to mediate calcium release from the ER when it is activated by calcium entry through the cytomembrane, a mechanism called calcium-induced calcium release (64, 65).

    cAMP-PKA signaling pathway is required for ADM-induced increases in [Ca2+]i and NO release

    ADM elevates intracellular cAMP levels in various types of cells (1, 18), including neural cells (66, 67), and the cAMP-PKA signaling pathway has been shown to mediate many of ADM’s effects, including cell proliferation and migration (68, 69), cell protection (70), and cell regeneration (71). Consistent with previous reports (66, 67), our results show that ADM increases intracellular cAMP levels in SK-N-SH cells, suggesting that the cAMP-PKA signaling pathway is recruited by ADM. Interestingly, ADM tends to increase intracellular cAMP levels 10 min after ADM treatment, and significant increases in cAMP levels occur after 20 min. Because the ADM-induced increases in cAMP occur slightly earlier than the increases in [Ca2+]i and NO release, our data suggest that ADM-induced recruitment of the cAMP-PKA signaling pathway precedes and mediates ADM-induced effects on [Ca2+]i and NO release. This mechanism is indeed confirmed by the observations that a cAMP analog stimulated comparable levels of NO release as ADM and that the ADM-induced increases in [Ca2+]i and NO release were abolished by H-89, a PKA inhibitor. Interestingly, cAMP and its associated signaling pathway have been reported to increase NO production via calcium-dependent mechanisms in urothelial cells (72) and enterocytes (73).

    The mechanisms by which the cAMP-PKA signaling pathway mediates ADM-induced effects on [Ca2+]i and NO release remain to be elucidated. It has been shown that the cAMP-PKA pathway increases L-type VDCC activities in various cell types (72, 74, 75), including neurons (76, 77). Therefore, the cAMP-PKA pathway may mediate ADM’s effects by increasing calcium influx via the L-type VDCC. Alternatively, the cAMP-PKA may also potentiate RyR-mediated calcium release, because PKA has been shown to lead to phosphorylation of RyR and consequent increases in calcium release from the ER in cardiac myocytes (78). Moreover, PKA may increase calcium release from the ER by activating ER-associated calcium-ATPase and thus enhancing ER calcium load (79).

    Summary

    The present study demonstrates that ADM stimulates NO release from human neuroblastoma cells. Furthermore, we have identified the mechanisms involved in this effect. Thus, binding of ADM to its receptors stimulates calcium influx through the L-type VDCC; the rise in [Ca2+]i is also amplified by calcium-induced calcium release from the ER via the RyR. Concentrated intracellular calcium then leads to activation of neuronal NOS, which, in turn, results in synthesis and release of NO. Although the mechanisms by which ADM activates the L-type VDCC have not been directly addressed in the present study, we suggest that the cAMP-PKA pathway, which is required for the ADM-induced responses, contributes to activation of the calcium channel. This mechanism together with the possibility that the cAMP-PKA pathway mediates ADM’s effects by stimulating activity of the RyR and/or ER-associated calcium-ATPase remain to be clarified (Fig. 9).

    The development of potential ADM therapies for neurological disorders has been hampered by a lack of information about the molecular mechanisms for ADM’s effects in the brain. By characterizing the effects of ADM on [Ca2+]i and NO and by providing insight into molecular mechanisms for ADM-induced events in the neuroblastoma cells, our data not only contribute to the fundamental understanding about interactions among ADM, calcium, and NO in neurons, but also provide a theoretical basis for potential therapeutic applications of ADM in the CNS.

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