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the Center for Neuroscience, National Sun Yat-sen University (S.H.H.C.), Kaohsiung
Department of Pharmacology, National Cheng Kung University (K.S.H., C.C.H.), Tainan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital (L.L.W., C.C.O., J.Y.H.C.), Kaohsiung, Taiwan, Republic of China.
Abstract
The rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a central site via which angiotensin II (Ang II) elicits its pressor effect. We tested the hypothesis that NADPH oxidase-derived superoxide anion (O2·eC) in the RVLM mediates Ang IIeCinduced pressor response via activation of mitogen-activated protein kinase (MAPK) signaling pathways. Bilateral microinjection of Ang II into the RVLM resulted in an angiotensin subtype 1 (AT1) receptor-dependent phosphorylation of p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), in the ventrolateral medulla. The Ang IIeCinduced p38 MAPK or ERK1/2 phosphorylation was attenuated by application into the RVLM of a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), an antisense oligonucleotide that targets against p22phox or p47phox subunit of NADPH oxidase mRNA, or the superoxide dismutase mimetic tempol. DPI or antisense p22phox or p47phox oligonucleotide treatment also attenuated the AT1 receptor-dependent increase in O2·eC production in the ventrolateral medulla elicited by Ang II at the RVLM. Functionally, Ang IIeCelicited pressor response in the RVLM was attenuated by DPI, tempol, or a p38 MAPK inhibitor, SB203580. The AT1 receptor-mediated enhancement of the frequency of glutamate-sensitive spontaneous excitatory postsynaptic currents induced by Ang II in RVLM neurons was also abolished by SB203580. These results suggest that NADPH oxidase-derived O2·eC underlies the activation of p38 MAPK or ERK1/2 by Ang II in the ventrolateral medulla. Furthermore, the p38 MAPK signaling pathway may mediate Ang IIeCinduced pressor response via enhancement of presynaptic release of glutamate to RVLM neurons.
Key Words: mitogen-activated protein kinases angiotensin II superoxide anion NADPH oxidase rostral ventrolateral medulla blood pressure
Introduction
In addition to its well-known actions on the vasculature, angiotensin II (Ang II) also plays a critical role in central regulation of circulatory functions.1,2 One brain target where Ang II exerts its influence on cardiovascular control is rostral ventrolateral medulla (RVLM),3,4 where premotor neurons that maintain tonic sympathetic vasomotor outflow are located.5 The RVLM contains a high density of Ang II receptors6 and is a major site of the sympathoexcitatory and pressor actions of the octapeptide.7,8 It is generally accepted that activation of Ang II type 1 (AT1) receptors in the RVLM contributes mainly to the cardiovascular effects of Ang II.9,10
The reactive oxygen species, particularly superoxide anion (O2·eC), is an important intracellular messenger for brain Ang II. The octapeptide increases the activity of NADPH oxidase, the major source of O2·eC in the vasculature,11 and enhances O2·eC production in the central nervous system.12,13 Intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the increase in renal sympathetic nerve activity or pressor response induced centrally by Ang II.13,14 In the brain, overexpression of superoxide dismutase (SOD), the enzyme responsible for O2·eC breakdown, also abolishes the central pressor effect of the octapeptide.12,15
A potential intracellular signaling pathway that mediates Ang IIeCelicited responses is for O2·eC to activate mitogen-activated protein kinases (MAPKs).16 At least 6 major mammalian MAPK subfamilies have been identified, of which p38 MAPK, extracellular signal-regulated protein kinase 1/2 (ERK1/2), and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) are the best characterized.17 Activation of MAPKs is involved in Ang IIeCpromoted cardiac hypertrophy18 or contractility of mesenteric resistance arteries19 in hypertensive rats. In vascular smooth muscle cells and cardiac myocytes, oxidative stress promoted by Ang II leads to activation of MAPKs.16,18eC20
The specific central site at which NADPH oxidase-derived O2·eC mediates pressor response to brain Ang II and the O2·eC-sensitive intracellular signaling pathway involved remain to be delineated. The present study evaluated the hypothesis that NADPH oxidase-derived O2·eC in the RVLM mediates Ang IIeCinduced pressor response via activation of MAPK signaling pathways. Our results demonstrate that activation of p38 MAPK by NADPH oxidase-derived O2·eC in the RVLM plays a critical role in the short-term AT1 receptor-mediated pressor response to Ang II, possibly via an enhancement of presynaptic release of glutamate to RVLM neurons.
Materials and Methods
A brief summary of our experimental strategies is provided. Detailed description of materials and methods can be found in the online data supplement available at http://circres.ahajournals.org.
Activation of MAPKs in Ventrolateral Medulla by Ang II
Adult male Sprague-Dawley rats (The Experimental Animal Center of the National Applied Research Laboratories, Taiwan) maintained under propofol anesthesia (30 mg·kgeC1·heC1) received bilateral microinjection of Ang II into the RVLM, given alone or together with the AT1 receptor antagonist losartan or the AT2 receptor antagonist PD123319. A total volume of 50 nL was delivered to each side of the RVLM over 1 to 2 minutes to allow for complete diffusion of the test agents. The ventrolateral medulla that contained bilateral RVLM was removed at various postinjection time intervals, and the expression of total or phosphorylated p38 MAPK, ERK1/2, or SAPK/JNK was determined by Western blot analysis.
Involvement of NADPH Oxidase or Superoxide in Ang IIeCInduced MAPK Phosphorylation in Ventrolateral Medulla
We ascertained a causative involvement of NADPH oxidase or O2·eC in Ang IIeCinduced MAPK phosphorylation in the ventrolateral medulla by bilateral comicroinjection of Ang II and the NADPH oxidase inhibitor diphenylene iodinium (DPI) or the SOD mimetic tempol into the RVLM. To further investigate the involvement of the p22phox or p47phox subunit of NADPH oxidase in Ang IIeCinduced MAPK phosphorylation, antisense (ASON) or sense (SON) p22phox or p47phox oligonucleotide was microinjected bilaterally into the RVLM 24 hours before Ang II administration. The effectiveness of ASON to block p22phox or p47phox mRNA expression was confirmed by real-time reverse-transcription polymerase chain reaction analysis.
Involvement of NADPH Oxidase in Ang IIeCInduced Superoxide Production in Ventrolateral Medulla
The causative role of NADPH oxidase in Ang IIeCinduced O2·eC production was examined by pretreatment with losartan or PD123319, NADPH oxidase inhibitors, apocynin (APO) or DPI, or p22phox or p47phox ASON or SON. Losartan, PD123319, APO, or DPI was comicroinjected with, and ASON or SON was microinjected 24 hours before, Ang II application. O2·eC production in the ventrolateral medulla was determined by the lucigenin-enhanced chemiluminescence method.
Angiotensin Receptor-Dependent Phosphorylation of p47phox Subunit of NADPH Oxidase in Ventrolateral Medulla
Serine phosphorylation of the p47phox subunit of NADPH oxidase induced by Ang II was determined by immunoprecipitation followed by immunoblotting, and the subtype of Ang II receptors involved was studied by bilateral comicroinjection of losartan or PD123319 into the RVLM.
Intracellular Signaling for Pressor Response to Ang II
Whether NADPH oxidase, O2·eC, or MAPK is engaged in pressor response to Ang II was studied by bilateral comicroinjection of the octapeptide with DPI, tempol, the p38 MAPK inhibitor SB230850, the ERK1/2 inhibitor U0126, or the JNK inhibitor SP600125 into the RVLM.
Cellular Mechanisms Underlying Activation of p38 MAPK in AT1 Receptor-Dependent Excitation of RVLM Neurons by Ang II
To further delineate the cellular mechanisms via which activation of p38 MAPK underlies the AT1 receptor-dependent pressor action of Ang II in the RVLM, we examined the effects of the octapeptide on spontaneous excitatory postsynaptic currents (sEPSCs) recorded from RVLM neurons, using the whole-cell patch-clamp technique in brain stem slice preparations. We also evaluated the effects of losartan, PD123319, and SB203580 on the Ang II actions.
Results
Selective Activation of MAPKs by Ang II in Ventrolateral Medulla
Bilateral microinjection of Ang II (50 pmol) into the RVLM resulted in activation of p38 MAPK or ERK1/2, but not SAPK/JNK, in the ventrolateral medulla (Figure 1A through 1C). Phosphorylation of p38 MAPK or ERK1/2 was detected 5 minutes after Ang II treatment, lasting 10 or 30 minutes, respectively (Figure 1D). The Ang IIeCinduced p38 MAPK (Figure 2A) or ERK1/2 (Figure 2B) phosphorylation was significantly antagonized by coadministration of losartan (2 nmol) but not PD123319 (2 nmol). Both Ang II antagonists, on the other hand, did not affect SAPK/JNK phosphorylation in the RVLM (Figure 2C). Furthermore, none of those treatments (Figure 1A through 1C and Figure 2A through C) elicited a discernible effect on total p38 MAPK, ERK1/2, or SAPK/JNK levels in the ventrolateral medulla.
Effects of DPI or Tempol on MAPK Activation by Ang II in Ventrolateral Medulla
Bilateral comicroinjection of DPI (1.5 nmol) or tempol (50 nmol) into the RVLM significantly blunted the peak activation of p38 MAPK (Figure 2A) or ERK1/2 (Figure 2B) by Ang II (50 pmol) in the ventrolateral medulla. DPI or tempol, on the other hand, elicited minimal effect on basal expression of MAPKs (Figure 2A through 2C) or SAPK/JNK phosphorylation (Figure 2C).
Effects of p22phox or p47phox ASON on Ang II Activation of p38 MAPK or ERK1/2 in Ventrolateral Medulla
Whereas application of p22phox or p47phox SON was ineffective, bilateral microinjection of p22phox or p47phox ASON (50 pmol) into the RVLM 24 hours before Ang II administration significantly attenuated p38 MAPK or ERK1/2 phosphorylation induced in the ventrolateral medulla (Figure 3A and 3B) by the octapeptide (50 pmol). Real time reverse transcription polymerase chain reaction analysis (Figure 3C) confirmed that ASON pretreatment selectively suppressed the mRNA expression of p22phox or p47phox, but not the p67phox or gp91phox subunit of NADPH oxidase, and that control SON pretreatment was ineffective. Pretreatment with p22phox or p47phox ASON also did not affect total p38 MAPK or ERK1/2 level in the RVLM (data not shown). In a separate series of control experiments, bilateral microinjection of Glu (2 nmol) into the RVLM did not influence p38 MAPK (Figure 3A) or ERK1/2 (Figure 3B) phosphorylation in the ventrolateral medulla.
Differential Effects of Losartan, PD123319, or Tempol on Ang IIeCInduced O2·eC Production in Ventrolateral Medulla
Figure 4A shows that microinjection of Ang II (50 pmol) bilaterally into the RVLM increased O2·eC production significantly at 5 minutes after injection, lasting 20 minutes. The peak O2·eC production induced by Ang II was significantly inhibited by coadministration of losartan (2 nmol) or tempol (50 nmol), but not PD123319 (2 nmol). Losartan, PD123319, and tempol alone exerted minimal alteration in basal O2·eC level in the RVLM.
Effects of p22phox or p47phox ASON or NADPH Oxidase Inhibitors on Ang IIeCInduced O2·eC Production in Ventrolateral Medulla
Ang IIeCinduced O2·eC production in the ventrolateral medulla was also attenuated by pretreatment with p22phox or p47phox ASON (50 pmol; Figure 4B). Control treatment of p22phox or p47phox SON (50 pmol) was ineffective. Comicroinjection of APO (2 nmol) or DPI (1.5 nmol) also significantly blunted Ang IIeCinduced O2·eC production in the ventrolateral medulla. APO and DPI, however, exerted no discernible effect on basal O2·eC level in the ventrolateral medulla.
Effects of Losartan and PD123319 on Ang IIeCInduced p47phox Phosphorylation in Ventrolateral Medulla
Immunoprecipitated p47phox subunit of NADPH oxidase in the ventrolateral medulla examined for serine phosphorylation showed significant elevation in phosphorylated p47phox that was detectable 5 minutes after bilateral microinjection of Ang II into the RVLM, lasting 20 minutes (Figure 4C). Both losartan (2 nmol) and PD123319 (2 nmol) attenuated Ang IIeCinduced p47phox phosphorylation, although the AT1 antagonist was more effective. Bilateral microinjection of Glu into the RVLM (2 nmol), on the other hand, did not promote p47phox phosphorylation.
Effects of NADPH Oxidase Inhibitor or SOD Mimetic on Ang IIeCInduced Pressor Response
Bilateral microinjection of Ang II (50 pmol) into the RVLM evoked a short-term pressor response that lasted for 10 minutes. This pressor response was significantly blunted by coadministration of DPI (1.5 nmol; Figure 5A), APO (2 nmol; data not shown), and tempol (50 nmol; Figure 5B), but not 3-CP (50 nmol; Figure 5C), a compound that is structurally similar to tempol but that exhibits low O2·eC scavenging capacity.21 Treatment with DPI, APO, or tempol, on the other hand, did not alter basal mean systemic arterial pressure (MSAP), heart rate, or the pressor response to bilateral microinjection of Glu into the RVLM (2 nmol; Figure 5A through 5C). In addition, microinjection of Ang II, alone or in combination with various test agents, into areas adjacent to the confines of the RVLM did not result in appreciable alterations in basal MSAP or heart rate (data not shown).
Effects of MAPK Inhibitors on Ang IIeCInduced Pressor Response
Bilateral coadministration of the p38 MAPK inhibitor SB203580 (500 nmol; Figure 6A), but not the ERK1/2 inhibitor U0126 (500 nmol; Figure 6B) or the JNK inhibitor SP 600125 (500 nmol; Figure 6C), into the RVLM significantly attenuated the Ang IIeCpromoted pressor effects. None of the MAPK inhibitors, however, exerted discernible effect on basal cardiovascular parameters or Glu (2 nmol)-induced pressor response (Figure 6A through 6C).
Effects of Losartan, PD123319, or p38 MAPK Inhibitor on Ang IIeCInduced Cellular Response in RVLM Neurons
We measured sEPSCs in the RVLM neurons that were voltage-clamped at eC70 mV and were pharmacologically isolated from spontaneous inhibitory currents by including bicuculline methiodide (10 eol/L) and strychnine hydrochloride (0.5 eol/L) in the perfusate. These sEPSCs were totally blocked by coapplication of CNQX (20 eol/L) plus D-APV (50 eol/L) in the perfusate (data not shown), confirming that they are glutamate receptor-mediated synaptic events. In 5 RVLM neurons tested, Ang II (0.5 eol/L) significantly increased the mean frequency of the sEPSCs from 2.33±0.18 to 4.38±0.17 Hz (Figure 7A and 7F), and significantly shifted the cumulative inter-event interval distribution of sEPSCs to shorter intervals (Figure 7D). Both amplitude histogram (Figure 7B1 and 7B2) and cumulative probability plots (Figure 7C), however, showed no significant effect of Ang II on the sEPSC amplitude. The mean amplitude of sEPSCs (6.98±0.23 pA) recorded in the presence of Ang II (0.5 eol/L) was comparable to that of sEPSCs (6.65±0.21 pA) recorded under baseline condition (Figure 7E). Pretreatment with losartan (5 eol/L) or SB203580 (1 eol/L), but not PD123319 (5 eol/L), significantly blocked the enhancement of Ang II (0.5 eol/L) on sEPSC frequency recorded from RVLM neurons (Figure 8B). None of these treatments elicited discernible effects on control frequency of sEPSC (data not shown), nor did they affect sEPSC amplitude (Figure 8A) under baseline conditions or after application of Ang II.
Discussion
It is well documented that Ang II participates in central cardiovascular control by eliciting sympathoexcitatory and pressor actions7,8 through activation of AT1 receptors in the RVLM.9,10 In the present study, we identified that a novel intracellular signaling mechanism that underlies this action of the octapeptide entails AT1 receptor-dependent increases in sEPSC frequency by activation of p38 MAPK via NADPH oxidase-derived O2·eC in the RVLM.
Our study provided the first biochemical demonstration that Ang II induces AT1 receptor-dependent phosphorylation of p38 MAPK or ERK1/2 in the ventrolateral medulla. Activation of p38MAPK and ERK1/2 by Ang II has been reported in mesenteric smooth muscle cells,19,22,23 aortae,18,24 or cardiomyocytes.18,24,25 Similar to previous reports,19,22 we found that the time course of Ang IIeCinduced p38 MAPK phosphorylation was discernibly different from that of ERK1/2. We interpret these temporally different expression profiles to suggest that different upstream signaling pathways exist in linking AT1 receptor activation to phosphorylation of p38 MAPK or ERK1/2 in the RVLM. In support of this interpretation, inhibition of the Src family of protein tyrosine kinase, which plays a key role in coupling cell surface receptors with the cytoplasmic signaling machinery,26 abrogates Ang IIeCinduced ERK1/2 activation but only partially reverses p38 MAPK activity in vascular smooth muscle cells.19 In contrast to observations18,22,24 in aortae or cardiomyocytes, we found that activation of AT1 receptors by Ang II did not induce SAPK/JNK phosphorylation in the ventrolateral medulla over 60 minutes. Because SAPK/JNK phosphorylation in vascular smooth muscle cells commences at 15 minutes and peaks 30 minutes after application of the octapeptide,22 the possibility that Ang IIeCinduced activation of SAPK/JNK manifests a slower time course in the RVLM than phosphorylation of p38 MAPK or ERK1/2 is deemed unlikely.
Another intriguing finding of the present study is the identified role for NADPH oxidase-derived O2·eC as a second messenger in the RVLM that mediates Ang IIeCinduced activation of p38 MAPK or ERK1/2 via AT1 receptors. Activation of NADPH oxidase is a multi-step process that is initiated by serine phosphorylation of the cytosolic regulatory p47phox subunit.27 After translocation to the membrane, the activated p47phox subunit associates with the membrane-bound gp91phox and p22phox subunits to bring forth enzymatic activity.28 In this regard, we have detected the presence of membrane-bound p22phox and gp91phox, or cytosolic p47phox and p67phox subunit mRNA in the ventrolateral medulla. More importantly, we demonstrated that phosphorylation of serine residues of p47phox induced by Ang II in the RVLM, which was blunted by losartan, manifested a temporal profile that correlated positively with O2·eC production induced by the octapeptide. Furthermore, pretreatment with p22phox or p47phox ASON in the bilateral RVLM reversed Ang IIeCinduced O2·eC production and p38 MAPK or ERK1/2 phosphorylation. It follows that by acting on the AT1 receptors in the RVLM, Ang II may initiate a signaling cascade that leads to phosphorylation of p38 MAPK or ERK1/2 mediated by NADPH oxidase-derived O2·eC after the induction of serine phosphorylation of the p47phox subunit.29 On the basis of results from vascular smooth muscle cells, the potential upstream cellular signals linking Ang II to p47phox phosphorylation in the RVLM include Rac,14 phospholipase D,30 protein kinase C,30 or Rho-kinase.31
A MAPK-dependent pathway has been demonstrated to be necessary for Ang IIeCinduced pressor response in the RVLM.32 Functional evaluations in the present study extended this demonstration by showing, for the first time, that O2·eC-sensitive activation of p38 MAPK in the RVLM plays an active role specifically in the short-term pressor response to Ang II. In primary cultured brain stem neurons, the octapeptide increases the neuronal firing rate by inhibition of the delayed rectifier potassium current,33 an action that is associated with an increase in NADPH oxidase activity and O2·eC production. NADPH oxidase-dependent O2·eC is also involved in Ang IIeCinduced increase in intracellular Ca2+ concentration in neurons.34 We further demonstrated that, at the cellular level, Ang II elicits excitatory actions on RVLM neurons by increasing the frequency, but not the amplitude, of sEPSCs. A change in the amplitude of sEPSCs is classically interpreted as a postsynaptic modification, whereas a change in their frequency points toward presynaptic mechanisms that increase the probability of transmitter release.35,36 Together with the observations that the sEPSCs were completely antagonized by N-methyl-D-aspartate and noneCN-methyl-D-aspartate antagonist, our results suggest that an enhancement of presynaptic release of glutamate may underlie the excitatory actions of Ang II on RVLM neurons. More importantly, this modulatory action is mediated by AT1 receptors and requires activation of p38 MAPK. The lack of effect of Ang II on the amplitude of sEPSCs also implies that synaptic modulation by the octapeptide on RVLM neurons is not mediated by a change in postsynaptic sensitivity to glutamate.
Whereas results from our biochemical experiments indicated that Ang II promoted phosphorylation of ERK1/2 in the RVLM via NADPH oxidase-derived O2·eC, those from our physiological experiments revealed that this MAPK member may not participate in the short-term pressor response to the octapeptide. ERK1/2 is a major growth-signaling kinase that regulates the activity of a number of transcription factors, leading to induction of downstream target genes encoding proteins for smooth muscle cell growth, proliferation, and/or migration.16,19 In this regard, ERK1/2-dependent, p38 MAPK-independent pathways activate the transcription factor AP-1, leading to upregulation of c-fos mRNA in vascular smooth muscle cells.19 It is therefore possible that Ang IIeCinduced ERK1/2 phosphorylation may function in central cardiovascular regulation via cellular events in the RVLM that are involved in transcription regulation. This possibility, however, requires further investigation.
Our results showed that peak p38 MAPK phosphorylation (Figure 3) or O2·eC generation (Figure 4) was detected at a time point during which Ang IIeCpromoted pressor response (Figure 5 and Figure 6) has already reached its peak. This seemingly contradicts our conclusion that activation of p38 MAPK by the NADPH oxidase-derived O2·eC in the RVLM mediates the pressor response to Ang II. This discrepancy essentially arises from technical differences between physiological and biochemical experiments. Whereas data collection in the former is in a continuous manner, the earliest feasible time point for collection of samples from the ventrolateral medulla is 5 minutes after application of test agents into the RVLM. Most importantly, substantiation of our conclusion is provided by the demonstration that SB 203580, DPI, and tempol elicited comparable suppression on Ang IIeCinduced pressor response in the RVLM (Figure 5 and Figure 6). Our control results with Glu additionally established that our observations are specific to the actions of Ang II in the RVLM.
We noted that compared with an almost complete blockade of Ang IIeCpromoted pressor response by the O2·eC scavenger (Figure 5B), NADPH oxidase inhibitor elicited only a partial reversal (Figure 5A). These observations suggest that O2·eC generated from other cellular sources, eg, mitochondria,37 may also participate in Ang IIeCpromoted cardiovascular responses in the RVLM. Ang IIeCinduced inhibition of the delayed rectifier potassium currents in cultured brain stem neurons is mediated by NADPH oxidase-dependent O2·eC, but not a hydrogen peroxide mechanism.33 This dampens the possibility that, as a product of degradation of O2·eC, hydrogen peroxide in the RVLM may also participate in our observed physiological and biochemical responses to Ang II at the RVLM. Acute microinjection of Ang II into the RVLM in the present study may also account for the lack of upregulation of almost all NADPH oxidase subunits seen after long-term (24 hours to 7 days) treatment with the octapeptide.30,31
In conclusion, the present study unveiled a novel signaling pathway for the short-term pressor response to Ang II that entails AT1 receptor-dependent activation of p38 MAPK via NADPH oxidase-derived O2·eC in the RVLM. We further showed that the excitatory action of Ang II engages an enhancement of presynaptic glutamate release to RVLM neurons, a synaptic modulation that is AT1 receptor- and p38 MAPK-dependent. Increased levels of brain Ang II and O2·eC in the RVLM are associated with sympathoexcitation during hypertension,38,39 acute stress,40 or chronic heart failure.13 Furthermore, inhibition of MAPKs in the RVLM normalizes arterial pressure in spontaneously hypertensive rats.32 Antagonism of O2·eC-dependent p38 MAPK signaling pathway may therefore represent a novel approach to counteract sympathoexcitation in cardiovascular disorders that are associated with enhanced brain Ang II.
Acknowledgments
This study was supported by research grants NSC-94eC2752-B-110eC001-PAE, NSC-94eC2752-B-110eC002-PAE (S.H.H.C.), and NSC94eC2752-B075-B-001-PAE (J.Y.H.C.) from National Science Council, and VGHKS 94eC052 (J.Y.H.C.) from Kaohsiung Veterans General Hospital, Taiwan, Republic of China.
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