Am J Physiol Cell Physiol 306: C736–C744, 2014. First published January 8, 2014; doi:10.1152/ajpcell.00131.2013.

Direct evidence of intracrine angiotensin II signaling in neurons Elena Deliu,1* G. Cristina Brailoiu,2* Satoru Eguchi,3 Nicholas E. Hoffman,4,5 Joseph E. Rabinowitz,1,5 Douglas G. Tilley,1,5 Muniswamy Madesh,4,5 Walter J. Koch,1,5 and Eugen Brailoiu1,5 1

Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania; 2Department of Pharmaceutical Sciences, Jefferson School of Pharmacy, Thomas Jefferson University, Philadelphia, Pennsylvania; 3 Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania; 4Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania; and 5Center for Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania Submitted 8 May 2013; accepted in final form 2 January 2014

Deliu E, Brailoiu GC, Eguchi S, Hoffman NE, Rabinowitz JE, Tilley DG, Madesh M, Koch WJ, Brailoiu E. Direct evidence of intracrine angiotensin II signaling in neurons. Am J Physiol Cell Physiol 306: C736 –C744, 2014. First published January 8, 2014; doi:10.1152/ajpcell.00131.2013.—The existence of a local renin-angiotensin system (RAS) in neurons was first postulated 40 years ago. Further studies indicated intraneuronal generation of ANG II. However, the function and signaling mechanisms of intraneuronal ANG II remained elusive. Since ANG II type 1 receptor (AT1R) is the major type of receptor mediating the effects of ANG II, we used intracellular microinjection and concurrent Ca2⫹ and voltage imaging to examine the functionality of intracellular AT1R in neurons. We show that intracellular administration of ANG II produces a dose-dependent elevation of cytosolic Ca2⫹ concentration ([Ca2⫹]i) in hypothalamic neurons that is sensitive to AT1R antagonism. Endolysosomal, but not Golgi apparatus, disruption prevents the effect of microinjected ANG II on [Ca2⫹]i. Additionally, the ANG II-induced Ca2⫹ response is dependent on microautophagy and sensitive to inhibition of PLC or antagonism of inositol 1,4,5-trisphosphate receptors. Furthermore, intracellular application of ANG II produces AT1R-mediated depolarization of hypothalamic neurons, which is dependent on [Ca2⫹]i increase and on cation influx via transient receptor potential canonical channels. In summary, we provide evidence that intracellular ANG II activates endolysosomal AT1Rs in hypothalamic neurons. Our results point to the functionality of a novel intraneuronal angiotensinergic pathway, extending the current understanding of intracrine ANG II signaling.

tion of ANG II, particularly in hypothalamic nuclei involved in cardiovascular control (22). An intracellular, nonsecreted form of renin is present in neurons and may be involved in intraneuronal synthesis of angiotensin peptides (22, 40). ANG II has been detected in the cell bodies and fibers of several hypothalamic nuclei (22, 40); association of ANG II with intraneuronal vesicles has also been reported (22, 40). AT1Rs are expressed in the hypothalamus (1, 33). AT1Rs may signal through different second messengers by coupling to different G proteins (15). Coupling of AT1Rs to Gq/11 proteins induces Ca2⫹dependent responses (42). Despite compelling evidence supporting the physiological and/or pathophysiological relevance of the brain RAS in cardiovascular control, thirst, memory facilitation, and metabolism (3, 40, 47), the functional significance of intracellular ANG II in neurons remains to be established. We and others have characterized effects mediated by intracellular ANG II in other tissues such as myometrium (17) and vascular smooth muscle cells (8, 25). Therefore, in the present study, we used intracellular microinjection and concurrent imaging to characterize intracellular ANG II-mediated signaling in hypothalamic neurons.1

calcium; endoplasmic reticulum; renin-angiotensin system

Ethical approval. Animal protocols were approved by the Institutional Animal Care and Use Committee. Chemicals. Bafilomycin A1, brefeldin A, ryanodine, and xestospongin C were obtained from EMD Millipore (Billerica, MA); ANG II, heparin, rapamycin, U-73122, SKF-96365, BAPTA-AM, 1-oleoyl2-acetyl-sn-glycerol (OAG), and CV-11947 (candesartan) from SigmaAldrich (St. Louis, MO); and Ned-19 and PD-123319 ditrifluoroacetate from Tocris Bioscience (R & D Systems, Minneapolis, MN). Rat AT1R in pIRES2 DsRed-Express2 vector (AT1R-DsRed) plasmid cDNA and control empty vector (DsRed) were provided by Dr. Satoru Eguchi. To create mammalian expression, the vector encoding rat AT1aR-hemagglutinin and independent red fluorescent protein, the polymerase chain reaction product of rat AT1aR cDNA fused with NH2-terminal hemagglutinin (18) in pcDNA3 vector (kindly provided by Dr. Laszlo Hunyady), was ligated into pIRES2 DsRed-Express2 vector (Clontech, Mountain View, CA). DNA constructs and transfection. U2OS cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM (Mediatech, Herndon, VA) containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). U2OS cells were transiently transfected with DsRed and DsRed-tagged rat AT1R (AGTRA_RAT, primary accession no. P25095) using TurboFectin 8.0 according to the

THE RENIN-ANGIOTENSIN SYSTEM

(RAS) exists not only as a circulating hormonal system regulating blood pressure and water/electrolyte homeostasis, but also as a local system, with important physiological/pathophysiological implications in several organs, including the brain (32, 47). In addition, increasing evidence supports the existence of an intracellular RAS, converging to intracellular synthesis and action of ANG II (14, 29). Whether systemic, local, or intracellular, the central effector peptide of RAS is ANG II, formed by sequential cleavage of angiotensinogen by renin and angiotensin-converting enzyme/chymase. The ANG II type 1 receptor (AT1R) is the main G protein-coupled receptor (GPCR) mediating the effects of ANG II (15). With respect to neurons, increasing evidence supports neuronal synthesis of angiotensinogen and intraneuronal genera-

* E. Deliu and G. C. Brailoiu contributed equally to this work. Address for reprint requests and other correspondence: E. Brailoiu, Center for Translational Medicine, Temple Univ. School of Medicine, 3500 N. Broad St., Rm. 951, Philadelphia, PA 19140 (e-mail: [email protected]). C736

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This article is the topic of an Editorial Focus by Robert Wondergem (45a).

0363-6143/14 Copyright © 2014 the American Physiological Society

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Table 1. Control experiments ⌬[Ca2⫹]i, nM

⫺8

ANG II (10 M) Control buffer CV-11947 (10⫺7 M) PD-123319 (10⫺7 M)

Untransfected U2OS cells

DsRed-transfected U2OS cells

17 ⫾ 6 24 ⫾ 4

28 ⫾ 5 22 ⫾ 6

AT1R-transfected U2OS cells

Primary hypothalamic neurons

25 ⫾ 4 23 ⫾ 5

57 ⫾ 4 32 ⫾ 4

Values are means ⫾ SE. Changes in intracellular Ca2⫹ concentration (⌬[Ca2⫹]i) produced by ANG II, CV-11947, and PD-123319 in untransfected U2OS cells, DsRed-transfected U2OS cells, ANG II type 1 receptor (AT1R)-transfected U2OS cells, or hypothalamic neurons were negligible.

manufacturer’s protocol (OriGene Technologies, Rockville, MD). Cells were used 24 – 48 h after transfection. Neuronal cell culture. Hypothalamic neurons were dissociated from neonatal (1- to 2-day-old) Sprague-Dawley rats (Ace Animal, Boyertown, PA) of both sexes, as previously described (7). Newborn rats were decapitated, and brains were quickly removed surgically and immersed in ice-cold Hanks’ balanced salt solution (HBSS; Mediatech). The hypothalamus was identified, removed, minced, and subjected to enzymatic digestion (papain, 5 min, 37°C) and mechanical trituration in the presence of total medium [Neurobasal-A (Invitrogen, Carlsbad, CA) containing 1% GlutaMAX (Invitrogen), 2% penicillinstreptomycin-amphotericin B solution (Mediatech), and 10% fetal bovine serum]. Cells were cultured on round 25-mm glass coverslips coated with poly-L-lysine (Sigma-Aldrich) in six-well plates. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. The mitotic inhibitor cytosine ␤-arabinofuranoside (1 ␮M; Sigma-

Aldrich) was added to the culture on day 3 to inhibit glial cell proliferation. Immunocytochemistry and confocal imaging studies. At 48 h after hypothalamic neurons were transiently transfected with DsRed-tagged rat AT1R and green fluorescent protein (GFP)-Rab7 (Addgene, Cambridge MA) using TurboFectin 8.0 according to the manufacturer’s protocol, they were fixed with 4% paraformaldehyde, washed in PBS, and mounted with Dapi-Fluoromont-G (Southern Biotech, Birmingham, AL). Cells were imaged using a two-photon confocal microscope (model 710, Carl Zeiss) with a ⫻63 oil-immersion objective and ⫻1 digital zoom, with excitations set for 4=,6-diamidino-2phenylindole, GFP, and DsRed at 405, 488, and 561 nm, respectively. Images were analyzed using ZEN 2010 (Zeiss), as previously reported (28, 49). Ca2⫹ imaging. Intracellular Ca2⫹ concentration ([Ca2⫹]i) was measured as previously described (7, 9, 16, 17, 49). Cells were

Fig. 1. Microinjection of ANG II elevates cytosolic Ca2⫹ concentration ([Ca2⫹]i) in U2OS cells expressing ANG II type 1 receptor (AT1R). A: averaged traces (n ⫽ 6) of Ca2⫹ responses to ANG II (10⫺9–10⫺7 M), ANG II ⫹ the AT1R antagonist CV-11947 (10⫺7 M), and ANG II ⫹ the AT2R antagonist PD-123319 (10⫺7 M). Arrows indicate time of injection. B: increases in [Ca2⫹]i produced by 10⫺9–10⫺7 M ANG II in the absence and presence of AT1R or AT2R antagonist. *P ⬍ 0.00001 vs. control injections. #P ⬍ 0.00001 vs. 10⫺8 M ANG II. C–F: representative images showing AT1RDsRed fluorescence and fura 2-AM ratio of fluorescence (F340/ F380) before, during, and 6 min after microinjection of control buffer, ANG II (10⫺8 M), ANG II ⫹ CV-11947, or ANG II ⫹ PD-123319. Arrows indicate injected cells. Fluorescence scale (0 –3) is shown in C.

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incubated with 5 ␮M fura 2-AM (Invitrogen) in HBSS at room temperature for 45 min in darkness, washed three times with dye-free HBSS, and then incubated for 45 min to allow complete deesterification of the dye. Coverslips (25-mm-diameter) were subsequently mounted in an open-bath chamber (model RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope (Eclipse TiE, Nikon, Melville, NY) equipped with a Perfect Focus System and a CoolSnap HQ2 charge-coupled device camera (Photometrics, Tucson, AZ). During the experiments, the Perfect Focus System was activated. Fura 2-AM fluorescence (510-nm emission), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR 3.1 software (Nikon). The ratio of the fluorescence signal at 340 nm to that at 380 nm was converted to Ca2⫹ concentration (24). In Ca2⫹-free experiments, CaCl2 was omitted. Intracellular microinjection. Intracellular microinjections were performed using FemtotipsII, InjectMan NI 2, and FemtoJet systems (Eppendorf), as described elsewhere (9, 16, 17, 49). Pipettes were backfilled with an intracellular solution containing (in mM) 110 KCl, 10 NaCl, and 20 HEPES (pH 7.2) or the compounds to be tested. Injection time was 0.4 s at 60 hPa with a compensation pressure of 20 hPa to maintain the microinjected volume to ⬍1% of cell volume, as measured by microinjection of a fluorescent compound (fura 2-free acid). The intracellular concentration of chemicals was determined on the basis of the concentration in the pipette and the volume of injection. The cells to be injected were Z-scanned before injection, and the cellular volume was automatically calculated using NISElements AR 3.1 software. Measurement of membrane potential. The relative changes in membrane potential (Vm) of single neurons were evaluated using

bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)], a slow-response voltage-sensitive dye, as previously described (49). Upon membrane hyperpolarization, the dye concentrates in the cell membrane, leading to a decrease in fluorescence intensity, while depolarization induces sequestration of the dye into the cytosol, resulting in an increase in the fluorescence intensity. Cultured hypothalamic neurons were incubated for 30 min in HBSS containing 0.5 mM DiBAC4(3), and fluorescence was monitored at 0.17 Hz and 480-nm excitation/540-nm emission. Calibration of DiBAC4(3) fluorescence following background subtraction was performed using the Na⫹-K⫹ ionophore gramicidin in Na⫹-free physiological solution and various concentrations of K⫹ (to alter Vm) and N-methylglucamine (to maintain osmolarity). Under these conditions, Vm was approximately equal to the K⫹ equilibrium potential determined by the Nernst equation. The intracellular K⫹ and Na⫹ concentrations were assumed to be 130 and 10 mM, respectively. Data analysis. Values are means ⫾ SE. One-way ANOVA followed by post hoc Bonferroni’s and Tukey’s tests was used to assess significant differences between groups. P ⬍ 0.05 was considered statistically significant. RESULTS

Similar to previous reports (51), to ensure that the effects of microinjected ANG II are not mediated by cell-surface receptors, in all series of experiments, plasma membrane AT1Rs and AT2Rs were blocked by bath application of CV-11947 and PD-123319 (both 10⫺7 M), respectively. Control experiments. Microinjection of control buffer or ANG II (10⫺8 M) produced small, insignificant increases in

Fig. 2. Intracellular administration of ANG II increases [Ca2⫹]i in cultured hypothalamic neurons. A: averaged traces illustrating neuronal [Ca2⫹]i responses to microinjected ANG II (10⫺9–10⫺7 M), ANG II ⫹ CV-11947 (10⫺7 M), ANG II ⫹ PD-123319 (10⫺7 M), and ANG II ⫹ the Ca2⫹ chelator BAPTA-AM (200 ␮M). B: amplitude of [Ca2⫹]i increases produced by 10⫺9–10⫺7 M ANG II, ANG II ⫹ CV-11947, ANG II ⫹ PD-123319, and ANG II ⫹ BAPTA-AM. *P ⬍ 0.00001 vs. control injections. #P ⬍ 0.00001 vs. 10⫺8 M ANG II. C–F: representative fluorescence images of fura 2-AM-loaded hypothalamic neurons before (basal), during (injection), and 6 min after intracellular administration of control buffer, ANG II (10⫺8 M), ANG II ⫹ CV-11947, or ANG II ⫹ PD-123319. Arrows indicate injected cells. Fluorescence scale (0 –3) is shown in C. AJP-Cell Physiol • doi:10.1152/ajpcell.00131.2013 • www.ajpcell.org

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Fig. 3. Functional AT1Rs are localized to endolysosomes in hypothalamic neurons. A: ANG II-induced Ca2⫹ response is abolished after 1 h of incubation with bafilomycin A1 (Baf, 1 ␮M) or rapamycin (Rap, 30 ␮M), but not brefeldin (Bref, 10 ␮M). Averaged traces from 6 experiments are shown. B: increases in [Ca2⫹]i produced by microinjected ANG II in the absence or presence of Bref, Baf, or Rap. *P ⬍ 0.00001 vs. ANG II alone. C: AT1R is colocalized with Rab7, an endolysosomal marker, in hypothalamic neurons. Nuclei were labeled with 4=,6-diamidino-2-phenylindole (DAPI).

[Ca2⫹]i in untransfected U2OS cells. Similarly, in DsRedtransfected U2OS cells, intracellular administration of control buffer or ANG II had no effect. Also, microinjection of the AT1R antagonist CV-11947 or the AT2R antagonist PD123319 (both 10⫺7 M) did not elicit significant [Ca2⫹]i increases in AT1R-transfected U2OS cells or neurons. Six cells were injected in each of the above-mentioned control experiments. Data are presented in Table 1. Intracellular injection of ANG II increases [Ca2⫹]i in AT1Rtransfected cells. Microinjection of control buffer produced a small and insignificant [Ca2⫹]i elevation of 27 ⫾ 5 nM in AT1R-transfected U2OS cells, whereas intracellular administration of ANG II (10⫺9, 10⫺8, and 10⫺7 M final intracellular concentrations) dose dependently increased [Ca2⫹]i by 258 ⫾ 11, 711 ⫾ 8, and 972 ⫾ 17 nM, respectively (n ⫽ 6 cells for each concentration; Fig. 1, A–D). The rise in [Ca2⫹]i was abolished when ANG II (10⫺8 M) was coinjected with the AT1R antagonist CV-11974 (10⫺7 M, ⌬[Ca2⫹]i ⫽ 18 ⫾ 5 nM, n ⫽ 6) but was basically unaltered upon coadministration of the AT2R antagonist PD-123319 (10⫺7 M, ⌬[Ca2⫹]i ⫽ 697 ⫾ 7 nM, n ⫽ 6; Fig. 1, A, B, E, and F). Neither antagonist alone had an effect on its own when injected into AT1R-transfected U2OS cells (Table 1). Intracellular injection of ANG II activates AT1R in hypothalamic neurons. Microinjection of control buffer did not significantly elevate [Ca2⫹]i in cultured hypothalamic neurons (⌬[Ca2⫹]i ⫽ 22 ⫾ 4 nM, n ⫽ 19 neurons; Fig. 2, A–C), while intracellular administration of ANG II (10⫺9, 10⫺8, and 10⫺7 M final intracellular concentrations) produced fast, transient, and dose-dependent increases in [Ca2⫹]i: 197 ⫾ 6 nM (n ⫽ 6 of 34), 398 ⫾ 7 nM (n ⫽ 6 of 29), and 605 ⫾ 9 nM (n ⫽ 6 of 32), respectively (Fig. 2, A–D). The percentages of neurons responding to increasing concentrations of ANG II by elevated [Ca2⫹]i were 17.6%, 20.7%, and 18.75%, respectively, indicating that functional intracellular AT1R expression is restricted to a subpopulation of hypothalamic neurons.

Treatment with CV-11947 (10⫺7 M), an AT1R antagonist, but not PD-123319 (10⫺7 M), an AT2R antagonist, prevented the Ca2⫹ response to microinjected ANG II (10⫺8 M): ⌬[Ca2⫹]i ⫽ 17 ⫾ 5 nM (n ⫽ 22 of 22 neurons) in the presence of CV-11947 and 391 ⫾ 8 nM (n ⫽ 6 of 31) in the presence of PD-123319 (Fig. 2, A, B, E, and F). Pretreatment of neurons with the fast Ca2⫹ chelator BAPTA-AM (200 ␮M, 30 min) abolished the effect of ANG II (10⫺8 M) microinjection (⌬[Ca2⫹]i ⫽ 33 ⫾ 5 nM, n ⫽ 63 of 63; Fig. 2, A and B). Endolysosomal location of functional intracellular AT1R in neurons. Disruption of the Golgi apparatus by 1 h of incubation with brefeldin A (10 ␮M) (5) did not significantly affect the ANG II (10⫺8 M)-induced Ca2⫹ response of hypothalamic neurons (⌬[Ca2⫹]i ⫽ 411 ⫾ 9 nM, n ⫽ 6 of 36 neurons; Fig. 3, A and B). Nevertheless, the ANG II effect was largely abolished by 1 h of incubation with bafilomycin A1 (1 ␮M), a V-type ATPase inhibitor that prevents lysosomal acidification (6), or rapamycin (30 ␮M), an agent that blocks microautophagy (30): ⌬[Ca2⫹]i ⫽ 28 ⫾ 4 nM (n ⫽ 17 of 17) in neurons pretreated with bafilomycin A1 and 26 ⫾ 6 nM (n ⫽ 33 of 33) in neurons pretreated with rapamycin (Fig. 3, A and B). Endolysosomal location of AT1R in neurons was confirmed by colocalization of AT1R-DsRed with Rab7-GFP, a small GTPase associated with endosomes and lysosomes (45) (Fig. 3C). Activation of endolysosomal AT1R in neurons mobilizes Ca2⫹ from inositol 1,4,5-trisphosphate-dependent stores. In hypothalamic neurons incubated with Ca2⫹-free HBSS, ANG II (10⫺8 M) produced a robust increase in [Ca2⫹]i, with an amplitude of 283 ⫾ 9 nM (n ⫽ 6 of 35; Fig. 4A). This effect was slightly, but significantly, reduced compared with the amplitude of the same treatment in Ca2⫹-containing saline (398 ⫾ 7 nM, n ⫽ 6 of 29 neurons; Fig. 4A), indicating mobilization of extra- and intracellular Ca2⫹ pools by ANG II. The areas under the curve of the ANG II-induced responses in the presence and absence of extracellular Ca2⫹ in hypotha-

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Fig. 4. ANG II mobilizes Ca2⫹ from intracellular and extracellular pools. A–C: amplitudes (A), areas under the curve (B), and half-lives (T½, C) of Ca2⫹ responses to ANG II (10⫺8 M) in Ca2⫹-containing and Ca2⫹-free saline. *P ⬍ 0.00001 vs. Ca2⫹-containing saline.

lamic neurons were also distinct, albeit the difference was more pronounced (626 ⫾ 26 and 368 ⫾ 11, respectively, Fig. 4B); the half-lives were largely identical (Fig. 4C). The ANG II-dependent Ca2⫹ response in Ca2⫹-free saline was abolished in the presence of rapamycin (⌬[Ca2⫹]i ⫽ 23 ⫾ 4 nM, n ⫽ 23 of 23), the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) antagonists heparin and xestospongin C (⌬[Ca2⫹]i ⫽ 21 ⫾ 6 nM, n ⫽ 18 of 18), or the PLC inhibitor U-73122 (⌬[Ca2⫹]i ⫽ 24 ⫾ 5 nM, n ⫽ 26 of 26; Fig. 5, A and B). Incubation with ryanodine (10 ␮M), an antagonist of the ryanodine receptor, or Ned-19, which prevents Ca2⫹ release from acidic-like Ca2⫹ stores (37), did not significantly affect the ANG II-induced increase in [Ca2⫹]i: ⌬[Ca2⫹]i ⫽ 285 ⫾ 4 nM (n ⫽ 6 of 34) after ryanodine and 281 ⫾ 6 nM (n ⫽ 6 of 31) after Ned-19 (Fig. 5, A and B). Hypothalamic neurons depolarize upon stimulation of endolysosomal AT1R. Mean resting Vm in cultured hypothalamic neurons was ⫺52.9 ⫾ 0.1 mV (n ⫽ 173). Microinjection of control buffer did not modify the neuronal Vm (⌬Vm ⫽ 0.1 ⫾ 0.1 mV, n ⫽ 37), whereas intracellular administration of ANG II (10⫺8 M) depolarized 19% of hypothalamic neurons, with a mean amplitude of 3.74 ⫾ 0.14 mV (n ⫽ 6 of 31; Fig. 6). When the AT1R antagonist CV-11947 (10⫺7 M) was coinjected with ANG II, the effect of ANG II was abolished (⌬Vm ⫽ 0.3 ⫾ 0.11 mV, n ⫽ 34 of 34). Conversely, coadministration of the AT2R antagonist PD-123319 (10⫺7 M) did not affect the ANG II-induced depolarization (⌬Vm ⫽ 3.67 ⫾ 0.09 mV, n ⫽ 6 of 32; Fig. 6). Moreover, inhibition of microautophagy with rapamycin abolished the depolarization produced by ANG II microinjection in hypothalamic neurons (⌬Vm ⫽ 0.27 ⫾ 0.2 mV, n ⫽ 39 of 39; Fig. 6). Pretreatment of neurons for 30 min with BAPTA-AM (200 ␮M) prevented the effect of ANG II (⌬Vm ⫽ 0.27 ⫾ 0.15 mV, n ⫽ 71 of 71; Fig. 6), indicating that the increase in [Ca2⫹]i is a mandatory step in the ANG II-mediated depolarization. In the presence of SKF96365 (2 ␮M), which blocks receptor- and store-operated Ca2⫹ entry via transient receptor potential canonical (TRPC) channels (13, 50), the response to ANG II was also abolished (⌬Vm ⫽ 0.21 ⫾ 0.11 mV, n ⫽ 94 of 94; Fig. 6). Expression of several subtypes of TRPC channels has been documented in the human and rodent hypothalamus (19, 39). SKF-96365 (2 ␮M) did not block the Ca2⫹ elevation in response to KCl (30 mM; Fig. 7), which produces depolarization-induced activation of voltage-gated Ca2⫹ channels; thus, at the concentration tested in this study, SKF-96365 is devoid of inhibitory activity at voltage-gated Ca2⫹ channels. While

diacylglycerol (DAG) has been reported to promote TRPC channel activation downstream of PLC (44), in the present study, the membrane-permeable DAG analog OAG (100 ␮M) (43) had no effect on the resting Vm of hypothalamic neurons

Fig. 5. Activation of endolysosomal AT1R mediates Ca2⫹ release from inositol 1,4,5-trisphosphate (IP3)-activated pools. A: averaged Ca2⫹ responses of hypothalamic neurons to microinjected ANG II in Ca2⫹-free saline in the absence (0 Ca2⫹ alone) or presence of the microautophagy blocker rapamycin (Rap), inhibitors of Ca2⫹ stores [ryanodine (Ry), Ned-19, heparin, and xestospongin C (XeC)], or the PLC inhibitor U-73122. B: [Ca2⫹]i increases in response to intracellular administration of ANG II in Ca2⫹-free saline in the absence and presence of antagonists. *P ⬍ 0.00001 vs. ANG II alone.

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tion techniques to assess the role of intracellular ANG II in hypothalamic neurons. Initial experiments in AT1R-transfected U2OS cells confirm the validity of our methods and selectivity of receptor antagonists. Our results indicate that intracellular AT1R is functional in hypothalamic neurons; microinjection of ANG II produced a dose-dependent increase in [Ca2⫹]i mediated by AT1R activation. Roughly 20% of neurons were ANG II-sensitive, indicating that the response may be selective to discrete neuronal populations in hypothalamic nuclei. In an additional series of experiments, we examined the intracellular location of functional AT1R in hypothalamic neurons. We found that disruption of lysosomes, but not the Golgi apparatus, completely prevented the effect of microinjected ANG II. In the absence of reliable anti-AT1R antibodies (18, 26), we examined the distribution of AT1R and the endolysosomal-associated small GTPase Rab7 (45) in hypothalamic neurons and identified extensive colocalization, supporting the endolysosomal location of AT1R suggested by the functional studies. Increasing evidence indicates that GPCRs may signal from the endolysosomal compartment (9, 16, 17, 27). This is not surprising, since the endolysosomal membranes are organized into specialized domains where molecules can assemble into specific signaling complexes (23, 41). However, the binding pocket for ANG II is situated on the NH2-terminal side of the AT1R and, thus, within the endolysosomal lumen, a site that is

Fig. 6. Intracellular administration of ANG II depolarizes hypothalamic neurons. A: changes in membrane potential (Vm) of hypothalamic neurons in response to administration of control buffer, oleoyl-2-acetyl-sn-glycerol (OAG, 100 ␮M) or ANG II (10⫺8 M) alone, or ANG II ⫹ CV-11947, ANG II ⫹ PD-123319, ANG II ⫹ the microautophagy inhibitor rapamycin, ANG II ⫹ BAPTA-AM, or ANG II ⫹ the transient receptor potential canonical channel blocker SKF-96365. B: intracellular administration of ANG II (10⫺8 M) depolarizes hypothalamic neurons with an average amplitude of 3.74 ⫾ 0.14 mV (n ⫽ 6 of 31). Response was abolished by BAPTA-AM, SKF-96365, rapamycin, and CV-11947, but not by PD-123319. *P ⬍ 0.00001 vs. control inj. Application of membrane-permeant OAG had no effect on neuronal Vm (⌬Vm ⫽ 0.31 ⫾ 0.29 mV, n ⫽ 41 cells). inj, Intracellular microinjection; ex, extracellular administration.

(Fig. 6); as such, DAG is unlikely to be a mediator of ANG II-induced depolarization via TRPC channels. DISCUSSION

While the presence of an intracellular RAS in neurons has been proposed for four decades (20), the functional significance of intraneuronal ANG II remained unclear. The effects and signaling pathways of ANG II in the brain have been extensively studied (3, 40, 46, 47). The contribution of brain ANG II to central control of blood pressure, drinking behavior, and salt appetite is indisputable (46, 47); these effects are mainly AT1R-mediated (46, 47). In light of increasing evidence of the intracrine role of ANG II (14, 17), we used concurrent Ca2⫹-imaging and microinjec-

Fig. 7. SKF-96365 (2 ␮M) does not inhibit voltage-gated Ca2⫹ channels in hypothalamic neurons. A: averaged Ca2⫹ responses induced by 30 mM KCl in the absence and presence of SKF-96365 (2 ␮M). B: amplitudes of Ca2⫹ increases produced by KCl were 286 ⫾ 6.1 nM (n ⫽ 48 cells) in the absence and 279 ⫾ 6.7 nM (n ⫽ 57) in presence of SKF-96365. Areas under the curve were 246 ⫾ 7.2 and 243 ⫾ 6.9, respectively.

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not immediately accessible for peptidic ligands. Nevertheless, we previously demonstrated that ANG II (17) or endothelin-1 (16) applied to the cell cytosol can be readily transferred inside the endolysosomal vesicles via microautophagy, a constitutive process by which soluble substrates are directly engulfed (35). A similar mechanism seemed plausible in the case of neuronal lysosomes. Indeed, rapamycin, an inhibitor of the last stage of the microautophagic process (30), prevented the effect of intracellular ANG II in hypothalamic neurons. Interestingly, the response was very fast, while the microautophagic process is expected to occur rather slowly. The time necessary for microautophagy is largely dependent on the lysosomal membrane invagination step of the process, which occurs with a 20to 30-min lag; the subsequent vesicle scission step is very

rapid, occurring in seconds (30, 35). Given that microautophagy is an ongoing process, important in housekeeping and in maintenance of cytoplasmic mass (35), membrane invaginations are formed continuously, and cytoplasmic components may be rapidly taken up into the endolysosomal lumen. The microautophagic vesicles, as generated, contain cytoplasmic medium, which is expected to preserve ANG II unmodified. After vesicle scission, hydrolases act to break down the vesicle and release microautophagic components into the lysosomal lumen, while permeases, such as Atg22p, promote recycling of nutrients and energy (35). Next, we sought to characterize the signaling pathway triggered by activation of endolysosomal AT1R in hypothalamic neurons. Incubation of neurons in Ca2⫹-free saline reduced the

Endo-Lys

Microautophagy

ANG II

A 1R AT

ANG II

C

PL TRPC Ca2+

Na+

Ca2+

Na+ Na+ Na+ 2+ Ca 2+ Ca Ca2+

IP3

Ca2+ IP3R

Membrane depolarization ER

Fig. 8. Proposed mechanism of ANG II-induced activation of endolysosomal AT1R in hypothalamic neurons. Cytosolic ANG II activates AT1R located on the endolysosomes (Endo-Lys) upon microautophagy-dependent transfer to the vesicle lumen. This, in turn, stimulates phospholipase C (PLC) at the lysosomal membrane to produce IP3 and subsequent endoplasmic reticulum (ER)/lysosomal Ca2⫹ release via IP3 receptors (IP3Rs). Release of Ca2⫹ from the ER triggers activation of transient receptor potential canonical (TRPC) channels at the plasma membrane and Na⫹ and Ca2⫹ influx, resulting in membrane depolarization.

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amplitude and the area under the curve of the [Ca2⫹]i rise in response to intracellular ANG II, pointing to the occurrence of Ca2⫹ release and Ca2⫹ influx. IP3R blockade or microautophagy inhibition, but not inhibition of nicotinic acid adenine dinucleotide phosphate-dependent endolysosomal Ca2⫹ release or ryanodine receptor blockade, significantly reduced the neuronal Ca2⫹ response to microinjected ANG II, indicating that the endoplasmic reticulum (ER)- or lysosome-located IP3Rs (4, 21) are critically associated with endolysosomal AT1R signaling. AT1R couples to Gq/11-PLC to generate IP3 (15); this pathway is functional in hypothalamic neurons (42). In addition, PLC is expressed in the endolysosomal membrane (36); we found that PLC inactivation prevents the neuronal response to microinjected ANG II. Since ANG II modulates hypothalamic function via depolarization and/or increase in excitability of target neurons (2, 10, 34), we further assessed whether intracellular administration of ANG II affects Vm in cultured hypothalamic neurons. A subpopulation of tested neurons depolarized in response to microinjected ANG II; the response was AT1R-mediated and dependent on microautophagy. The percentage of neurons responding with a change in Vm correlated well with the proportion of neurons in which [Ca2⫹]i was elevated in response to ANG II microinjection. Whereas the mean amplitude of the depolarization triggered by microinjected ANG II is rather small (3.74 ⫾ 0.14 mV), it is largely similar to that elicited by the bath-applied peptide in the hypothalamic neurons in the paraventricular nucleus that project to the rostral ventrolateral medulla (11). This angiotensinergic pathway is involved in blood pressure regulation (47), endorsing a physiological relevance of the change in Vm observed upon endolysosomal AT1R activation. Changes in resting Vm determine the ease with which excitatory synaptic inputs bring Vm closer to the threshold for action potential firing. A ⬃4-mV depolarization of hypothalamic neurons in the paraventricular nucleus was associated with a fourfold increase in the firing frequency of action potential discharge in response to ANG II (11). Since DiBAC4(3) is a slow-response voltage-sensitive dye, fast changes in Vm and neuronal firing in response to ANG II microinjection may have been missed in the present study. ANG II has been shown to depolarize hypothalamic neurons of supraoptic or paraventricular nuclei via activation of nonselective cation channels (31, 48). Since mobilization of Ca2⫹ from intracellular stores is a prerequisite for the activation of nonselective cationic conductance (38), this appeared to be a likely explanation linking the two main events triggered by intracellular ANG II in cultured hypothalamic neurons. Indeed, we noticed that chelation of intracellular Ca2⫹ with BAPTA-AM abolished the Ca2⫹ response and the depolarization elicited by ANG II. Furthermore, blocking Ca2⫹ entry via the TRPC channel, a nonselective cation channel activated via PLC by Ca2⫹ release from the ER (12, 44) or directly by DAG (44), also abolished the depolarization. In our experimental paradigm, however, DAG seemed not to be involved in TRPC channel activation, since OAG, a DAG analog used to study the existence of DAG-sensitive currents (43), could not mimic ANG II-induced depolarization in hypothalamic neurons. Corroborating the findings of this study, we propose that, in hypothalamic neurons, intracellular ANG II may reach the AT1R in the endolysosomes by microautophagy. ANG II stimulates endolysosomal AT1R, leading to PLC activation and

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IP3-dependent Ca2⫹ release. The release of Ca2⫹ from the ER, in turn, activates nonselective cation channels, such as TRPC channels, at the plasma membrane, promoting cation entry and neuronal membrane depolarization. The proposed model is summarized in Fig. 8. Considering the plethora of effects elicited by ANG II in the hypothalamus (46, 47), our findings increase the understanding of intracrine ANG II signaling in neurons with implications for neuronal physiology. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grant HL-090804 (to E. Brailoiu). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. performed the experiments; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. analyzed the data; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. interpreted the results of the experiments; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. prepared the figures; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. drafted the manuscript; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. edited and revised the manuscript; E.D., G.C.B., S.E., N.E.H., J.E.R., D.G.T., M.M., W.J.K., and E.B. approved the final version of the manuscript; E.B. is responsible for conception and design of the research. REFERENCES 1. Allen AM, Moeller I, Jenkins TA, Zhuo J, Aldred GP, Chai SY, Mendelsohn FA. Angiotensin receptors in the nervous system. Brain Res Bull 47: 17–28, 1998. 2. Bai D, Renaud LP. ANG II AT1 receptors induce depolarization and inward current in rat median preoptic neurons in vitro. Am J Physiol Regul Integr Comp Physiol 275: R632–R639, 1998. 3. Baltatu OC, Campos LA, Bader M. Local renin-angiotensin system and the brain—a continuous quest for knowledge. Peptides 32: 1083–1086, 2011. 4. Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium 32: 235–249, 2002. 5. Betina V. Biological effects of the antibiotic brefeldin A (decumbin, cyanein, ascotoxin, synergisidin): a retrospective. Folia Microbiol (Praha) 37: 3–11, 1992. 6. Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85: 7972–7976, 1988. 7. Brailoiu E, Dun SL, Gao X, Brailoiu GC, Li JG, Luo JJ, Yang J, Chang JK, Liu-Chen LY, Dun NJ. C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro. Neuroscience 159: 492–500, 2009. 8. Brailoiu E, Filipeanu CM, Tica A, Toma CP, de Zeeuw D, Nelemans SA. Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta. Br J Pharmacol 126: 1133– 1138, 1999. 9. Brailoiu GC, Oprea TI, Zhao P, Abood ME, Brailoiu E. Intracellular cannabinoid type 1 (CB1) receptors are activated by anandamide. J Biol Chem 286: 29166 –29174, 2011. 10. Brown TM, McLachlan E, Piggins HD. Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons. Neuroscience 154: 839 –847, 2008. 11. Cato MJ, Toney GM. Angiotensin II excites paraventricular nucleus neurons that innervate the rostral ventrolateral medulla: an in vitro patchclamp study in brain slices. J Neurophysiol 93: 403–413, 2005. 12. Clapham DE. TRP channels as cellular sensors. Nature 426: 517–524, 2003. 13. Clapham DE, Julius D, Montell C, Schultz G. International Union of Pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels. Pharmacol Rev 57: 427–450, 2005.

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