Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis

Chuwen Li , Chao Zhang , Hefeng Zhou , Yu Feng , Fan Tang , Maggie P. M. Hoi , Chengwei He , Dan Ma , Chao Zhao , Simon M. Y. Lee. Frontiers in Molecu...
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Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway Sun Woo Jin, Chul Yung Choi , Yong Pil Hwang, Hyung Gyun Kim , Se Jong Kim, Young Chul Chung, Kyung Jin Lee, Tae Cheon Jeong, and Hye Gwang Jeong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05416 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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TOC graphic

STO-609 P CaMKK

Tetracaine

Compound C P

L-NAME

RyR Betulinic acid

AMPK

Ca2+

P

CaM W7

LTCC

P

CaMKII

Nifedifine KN-93

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eNOS

NO

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Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-

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Signaling Pathway

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Short title: Betulinic acid activates eNOS

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Sun Woo Jin †,¶ , Chul Yung Choi ‡,¶, Yong Pil Hwang §, Hyung Gyun Kim †, Se Jong Kim †,

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Young Chul Chung



, Kyung Jin Lee



, Tae Cheon Jeong #, *, Hye Gwang Jeong †, *

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College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea

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Jeollanamdo Institute of Natural Resources Research, Jeollanamdo 529-851, Republic of

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Korea

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§

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University of Korea, Jinju 660-759, Republic of Korea

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College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea

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#

Department of Pharmaceutical Engineering,



Department of Food Science, International

Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan

College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea

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The first two authors contributed equally to this work.

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* To whom correspondence should be addressed:

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Hye Gwang Jeong; College of Pharmacy, Chungnam National University, Daejeon 305-764,

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Republic

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[email protected]

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Tae Cheon Jeong: College of Pharmacy, Yeungnam University, Gyeongsan 712-749,

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Republic of Korea, Tel: +82-53-810-2819, E-mail: [email protected]

of

Korea,

Tel:

+82-42-821-5936,

Fax:

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+82-42-823-6566.

E-mail:

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ABSTRACT

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Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that attenuates vascular

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diseases and atherosclerosis, but the mechanism by which it stimulates endothelial nitric

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oxide synthase (eNOS) is unclear. eNOS is the key regulatory enzyme in the vascular

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endothelium. This study examined the intracellular pathways underlying the effects of BA on

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eNOS activity and endothelial nitric oxide (NO) production in endothelial cells. BA treatment

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induced both eNOS phosphorylation at Ser1177 and NO production. It also increased the

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level of intracellular Ca2+ and phosphorylation of Ca2+/calmodulin-dependent kinase IIα

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(CaMKIIα) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Inhibition of

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the L-type Ca2+ channel (LTCC) and the ryanodine receptor (RyR) abolished BA-induced

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intracellular levels of Ca2+ and eNOS phosphorylation. Treatment with W7 (a CaM

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antagonist), KN-93 (a selective inhibitor of CaMKII), and STO 609 (a selective inhibitor of

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CaMKK) suppressed eNOS phosphorylation and NO production. Moreover, AMP-activated

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protein kinase (AMPK) was induced by BA, and BA-induced eNOS phosphorylation was

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inhibited by compound C, an AMPK inhibitor. Taken together, these results indicate that BA

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activates

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Ca2+/CaMKK/AMPK pathways. These findings provide further insight into the eNOS

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signaling pathways involved in the anti-atherosclerosis effects of BA.

eNOS

phosphorylation

and

NO

synthesis

via

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Keywords: Betulinic acid; eNOS; AMPK; CaMKII; CaMKK

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the

Ca2+/CaMKII

and

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INTRODUCTION

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Endothelial activation and dysfunction play an important role in the maintenance of vascular

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integrity and homeostasis by regulating the bioavailability of endothelial nitric oxide (NO).1,2

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In the vascular endothelium, NO bioavailability is regulated mainly by endothelial NO

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synthase (eNOS) upon the conversion of l-arginine to l-citrulline, and it plays a protective

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physiological role.3 Therefore, activation of eNOS and subsequent NO production is

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considered a promising therapeutic approach for vascular diseases, including atherosclerosis.4

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Increases in intracellular free Ca2+ and calmodulin concentrations lead to eNOS activation

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and NO production, which requires the ubiquitous Ca2+-binding protein calmodulin.5,6 As a

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result of Ca2+-dependent eNOS activation, eNOS phosphorylation at Ser-1177 is regulated

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mainly by Ca2+/calmodulin-dependent kinase II (CaMK II).7 In addition, AMP-activated

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protein kinase (AMPK) has been reported to have an anti-atherosclerosis effect by promoting

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eNOS phosphorylation at Ser-1177, which is associated with activation and AMPK-induced

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phosphorylation stimulates NO release by endothelial cells.8,9,10 As a family of upstream

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AMPK kinases, AMPK phosphorylation is regulated mainly by Ca2+/calmodulin-dependent

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protein kinase kinase β (CaMKKβ) and liver kinase B1 (LKB1).11,12

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Betulinic acid (3 beta-hydroxylup-20(29)-en-28-oic acid) is a pentacyclic triterpene prepared

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from betulin obtained from white-barked birch trees.13,14,15 BA has been reported to show

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various types of pharmacological properties, including anti-inflammation,16 anticancer,17,18

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antimalarial,19 antiAIDS,20 antifatty liver,21 antidiabetes,22,23 antidepression,24 and antiplatelet

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activities. Also, BA showed potential effects on vasorelaxation, 26,27 and eNOS expression,

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involved in anti-atherosclerosis. However, the effect of BA on eNOS activity and its

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mechanism are unclear yet. In this study, we report the signaling pathways underlying the

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effects of BA on eNOS activity using EA.hy926 human endothelial cells. We found that BA

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induces eNOS phosphorylation on Ser1177 and NO production. Then, by investigating the

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effects of BA in the presence of various inhibitors of signaling pathways, we found that

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CaMK II and AMPK are involved in eNOS activation by BA. To the best of our knowledge,

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this is the first report describing calcium dependent eNOS phosphorylation by BA.

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MATERIALS AND METHODS

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Chemicals

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were

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purchased from Hyclone (Logan, UT), and 4,5-diaminofluorescein diacetate (DAF-2 DA)

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and fluo-4 acetoxymethyl ester (Fluo-4-AM) were purchased from Invitrogen (Carlsbad, CA).

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L-NAME, compound C, LY294002, PD98059, SB203580, SP600125, and N-[6-

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aminohexyl]-5-chloro-1-napthalenesulfonamide hydrochloride (W7) were purchased from

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Calbiochem (La Jolla, CA). STO-609 and KN-93 were purchased from Sigma-Aldrich (St.

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Louis, MO). Antibodies against p-eNOS, eNOS, p-AMPK, AMPK, p-CaMKIIα, p-CaMKKβ,

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and CaMKKβ as well as horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG

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antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin

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antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3-(4,5-

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Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB

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Corporation (Cleveland, OH). The cytotoxicity detection kit used to measure lactate

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dehydrogenase (LDH) release was purchased from Roche Applied Science (Indianapolis, IN).

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All other chemicals were of the highest grade commercially available.

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Cell culture

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EA.hy926 cells were obtained from the American Type Culture Collection (Bethesda, MD).

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The cells were grown in DMEM containing 10% FBS at 37°C in a humidified incubator with

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5% CO2. BA was dissolved in dimethylsulfoxide (DMSO) and stored at -20°C until use. The

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stock solution was prepared immediately before use. Control cells were added with DMSO

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alone and the final concentration of DMSO did not exceed 0.1%.

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Cytotoxicity of BA in endothelial cells

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Conventional MTT reduction and LDH assays were used to determine the toxicity of BA to

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EA.hy926. Cells were seeded in 48-well plates and incubated at 37°C for 24 h. Wells were

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treated with various concentration of BA and the plates were incubated at 37°C for 24 h.

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MTT solution was added, followed by incubation for 30 min, and formazan crystals were

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solubilized by adding DMSO. The absorbance at 550 nm was measured using a

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BioTek Synergy HT microplate reader (BioTek Instruments, Winooski). The media were

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assayed using an LDH assay at 490 nm with a BioTek Synergy HT microplate reader

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(BioTek Instruments, Winooski). Calculations of cell viability (%) and cytotoxicity (fold-

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change) were based on the absorbance of treated cells relative to that of cells exposed to

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DMSO alone.

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Western blot analysis

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After treatment, EA.hy926 cells were lysed in lysis buffer (120 mM NaCl, 40 mM Tris [pH

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8], and 0.1% NP40 [Nonidet P-40]) on ice for 30 min and centrifuged at 12,000 rpm for 20

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min. Supernatants were collected and protein concentrations were measured using a protein

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assay kit (Pro-Measure, Intron biotechnology). Aliquots of the lysates (50 µg protein) were

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boiled for 5 min and then electrophoresed in 10% SDS-PAGE gels. Then the proteins were

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transferred to PVDF membranes, which were incubated with primary antibodies. Then the

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membranes were incubated with secondary anti-mouse or anti-rabbit antibody. Finally, the

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protein bands were detected using an enhanced chemiluminescence Western blotting

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detection kit (Biofact). The integrated optical density for the protein band was calculated by

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Image-J software, and then the values were normalized to an internal control (β-actin level

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and/or total forms).

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Measurement of NO production

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The production of NO was measured using the NO-specific fluorescent dye DAF-2 DA

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(Calbiochem) as described previously.29 In brief, EA.hy926 cells were grown to 95%

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confluence in 96-well plates and serum-starved overnight. The cells were loaded with DAF-2

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DA (final concentration, 2 µM) for 30 min at 37°C, rinsed three times with DMEM. Then the

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cells were either treated with BA or not, as indicated in the figure legends. In some

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experiments, l-NAME (100 µM), compound C (10 µM), W7 (10 µM), or KN-93 (10 µM)

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was added 30 min before loading with DAF-2 DA. Then the absorbance of culture media was

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measured at 495/515 nm using a BioTek Synergy HT microplate reader (BioTek Instruments,

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Winooski). The cells were fixed in 5% paraformaldehyde for 5 min at 4°C and then

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visualized using an EVOS fluorescence microscope (Life Technologies).

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Ca2+ measurement

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Intracellular levels of Ca2+ were measured using Fluo-4AM according to the manufacturer’s

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instructions. Briefly, cells were plated in 96-well plates, loaded with 5 µM Fluo-4AM for 30

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min at 37°C, and then incubated in the dark for 30 min at 25°C. The cells were treated with

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BA as indicated in the figure legends. Fluo-4AM was excited at a wavelength of 488 nm and

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emissions were monitored at 512 nm. All captured images were analyzed as described

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previously.30 In brief, fluorescence images of the selected cells were captured using an

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EVOS fluorescence microscope (Life Technologies). All quantifications were performed

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using the Image J software.

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Statistical analysis

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All experiments were repeated at least three times. Results are reported as means ± SD.

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The significance of differences between mean values was analyzed using the Newman–Keuls

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test for multi-group comparisons. Statistical significance was accepted for p-values