Angiotensin II-Induced Generation of Reactive Oxygen Species Is Regulated by a Phosphatidylinositol 3-Kinase/L-Type Calcium Channel Signaling Pathway
Angiotensin II-Induced Generation of Reactive Oxygen Species Is Regulated by a Phosphatidylinositol 3-Kinase/L-Type Calcium Channel Signaling Pathway
Journal of Life Science. 2015. Feb, 25(2): 231-236
Copyright © 2015, Korean Society of Life Science
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : January 03, 2015
  • Accepted : February 02, 2015
  • Published : February 28, 2015
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About the Authors
서연, 진
정민, 하
영환, 김
혜선, 이
순식, 배

Angiotensin II (AngII) is an essential hormone that affects vascular physiology. For example, stimulation of vascular smooth muscle cells (VSMCs) rapidly induces vasoconstriction and results in the up-regulation of blood pressure. Chronic stimulation of VSMCs with AngII also results in hypertrophy. In this study, we confirmed an involvement of phosphatidylinositol 3-kinase (PI3K)-dependent calcium mobilization in AngII-induced generation of reactive oxygen species (ROS). Stimulation of rat aortic smooth muscle cells (RASMCs) with AngII significantly induced the generation of ROS in a dose- and time-dependent manner. AngII-induced generation of ROS was completely abolished by pharmacological inhibition of PI3K (with LY294002), but inhibition of the ERK signaling pathway had no effect. AngII-induced calcium mobilization was completely blocked by inhibition of PI3K, whereas inhibition of the ERK signaling pathway by PD98059 was ineffective. Depletion of extracellular calcium or inhibition of the L-type calcium channel by nifedipine completely blocked AngII-induced calcium mobilization. Depletion of extracellular calcium by EGTA and incubation of RASMCs with calcium-free medium both significantly blocked AngII-induced ROS generation. Inhibition of the L-type calcium channel also significantly blocked AngII-induced ROS generation. These results suggest that AngII-induced ROS generation is regulated by calcium mobilization, which, in turn, is modulated by a PI3K/L-type calcium channel signaling pathway.
The renin-angiotensin-aldosteron (RAA) system is an important regulator of blood pressure, blood flow, and electrolyte homeostasis. Angiotensin II (AngII) which is produced by sequential proteolytic cleavage of angiotensinogen plays a crucial role in the regulation of systemic arterial blood pressure. For example, arterial blood pressure is rapidly increased by AngII-induced vasoconstriction [22 - 23 ]. In addition to this acute role of AngII in vasoconstriction, chronic stimulation of vessel walls with AngII facilitates hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs) [2] .
Angiotensin receptor (ATR) comprises two distinct receptors including angiotensin type 1 receptor (AT 1 R) and angiotensin type 2 receptor (AT 2 R). These receptors are coupled with G proteins. For example, AT 1 R is coupled with G q or G 12/13 and subsequently activates phospholipase C (PLC) [18] . On the other hand, AT 2 R is coupled with Gs followed by activation of protein kinase A (PKA). Activation of AT 1 R by AngII results in intracellular calcium mobilization which binds to calmodulin and activates myosin light chain kinase (MLCK) [24] . On the other hand, activation of AT 2 R receptor by AngII results in the chronic vascular response such as proliferation, migration, and growth. Although PLC and PKA seems to be major signaling molecules for AngII, multiple signaling pathways such as tyrosine kinase, MAPKs, Akt, and redox-sensitive transcription factors are also involved in the AngII-dependent regulation of VSMC physiology [18 - 19] .
Reactive oxygen species (ROS) are highly reactive molecules that are generated during normal physiological process as well as under various stress conditions [14] . Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is one of the major sources for ROS generation in vasculature. It consists of a catalytic subunit (Nox1, Nox2, Nox3, Nox4, or Nox5) and regulatory subunit (p22phox, p47phox, p67phox, and Rac1) [7] . The generation of ROS, most notably superoxide and hydrogen peroxide, in VSMCs can be regulated by vasoactive hormones including AngII [8] . In addition, it has been reported that platelet-derived growth factor (PDGF) [9] .
Recently, it has been reported that activation of Nox1 plays pivotal role in AngII-induced generation of ROS [8] . Since AT 1 R is coupled with G q , activation of PLC and subsequent generation of inositol-trisphosphate (IP 3 ) and diacylglycerol (DAG) is important for the generation of ROS [18] . For instance, PKC is activated by DAG and calcium which is mobilized by IP 3 from endoplasmic reticulum. Activation of PKC leads to the phosphorylation of p47 phox which is a key regulatory subunit for Nox [15] .
Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is activated by many types of cellular stimuli and regulates fundamental cellular functions such as transcription, translation, proliferation, growth, and survival [1] . Binding of growth factors to their cognate 1receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR) stimulates class Ia and Ib PI3K isoforms, respectively. PI3K catalyzes the production of phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) at the cell membrane. The role of ROS in the regulation of signaling molecules has been unveiled so far. For example, generation of ROS is required for AngII-induced activation of Akt in VSMCs [18] . In addition, ROS modulates intracellular free calcium concentration which is a major determinant of AngII-dependent vascular contraction. Up-regulation of intracellular calcium concentration by ROS enhances expression of AT1R, which in turn further stimulates calcium signaling [11] . However, there is no direct evidence that shows the involvement of PI3K and calcium signaling in the generation of ROS. In the present study, we showed that PI3K-dependent mobilization of intracellular calcium plays critical role in AngII-induced generation of ROS.
Materials and Methods
- Materials
Dulbecco′s modified eagle medium (DMEM) culture medium, fetal bovine serum (FBS), trypsin EDTA, and antibiotics were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Fura-2AM was purchased from Invitrogen (Grand Island, NY, USA) and PI3K inhibitor (LY294002) and ERK inhibitor (PD98059) were obtained from Merk Millipore (Billerica, MA, USA). Calcium inhibitor (Nifedipine), DCFDA, and AngII were purchased from Sigma-Aldrich (St Louis, MO, USA). All other high quality reagents were purchased from Sigma-Aldrich unless otherwise indicated.
- Rat aortic smooth muscle cell (RASMC) preparation
Pathogen-free Sprague-Dawley rats (3 to 4 weeks old) were housed under diurnal lighting conditions and allowed unlimited access to feed and tap water. All animal experiments were performed under university animal welfare guidelines (PNU-2009-2008). Rat aortic smooth muscle cells (RASMCs) were isolated from the thoracic aorta by a tissue explanting method as described previously [5] . Briefly, sodium barbital-anesthetized rats were perfused with PBS for 5 min. Thoracic aorta was aseptically isolated and surrounding fat and connective tissues were discarded. Vessels were longitudinally cut and the lumen side was scraped with a razor blade to remove the intima. Vessels were fragmented into 3-5 mm lengths and explanted lumen side down on collagen-coated culture dishes. Tissue fragments were maintained in DMEM/10% FBS with changing of the medium every two days. After seven days of explanting, tissue fragments were discarded and sprouted RASMCs were collected and used (passage 2 to 4) for experiments.
- ROS generation assay
To measure ROS generation, RASMCs were grown in 48-well plates, after being starved for 12 hr, RASMCs were incubated with 2 ' ,7 ' -dichlorofluorescein diacetat (DCF-DA, 20 μM) for 1 hr and then stimulated with AngII (1 μM) for 15 min under the indicated conditions. Cells were washed three times with phosphate-buffed saline (PBS). Fluorescence was detected using a fluorescence microscope at 10× magnification (Axiovert200, Carl Zeiss, Jena, Germany). Pixel intensity in a field was measured by MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
- Measurement of cytosolic calcium concentration
Intracellular calcium concentration was measured using Fura-2/AM, a calcium-sensitive fluorescent dye, as described previously [3] . Briefly, a total of 1×10 6 RASMCs were incubated with 3 μM Fura-2/AM at 37℃ in fresh serum-free DMEM medium with stirring for 50 min. A total of 1×10 6 cells were aliquoted into Locke ' s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl 2 , 5 mM HEPES pH 7.3, 10 mM glucose, 2.2 mM CaCl 2 , and 0.2 mM EGTA). Fluorescence was measured at emission wavelength of 500 nm using and excitation wavelength of 340 nm and 380 nm.
- Statistical analysis
Data are expressed as means ± S.D. of three independent experiments ( n = 3 for each experiment). The statistical differences between treated and non-treated groups were analyzed by an unpaired Student ' s t -test to assess differences. P -values less than 0.05 were considered significant and indicated by *.
- AngII stimulates ROS generation in RASMCs
To ascertain capability of AngII in generation of ROS, we examined the effect of AngII on the generation of ROS in RASMCs. AngII (1 μM) significantly induced the generation of ROS in a time- and dose-dependent manner ( Fig. 1 ). Production of ROS reached a maximum within 15 min after stimulation ( Fig. 1A and 1B ). AngII evoked ROS generation at concentrations between 1 nM and 1 μM ( Fig. 1C and 1D ). These results indicate that AngII stimulates ROS generation in RASMCs.
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AngII-induced ROS generation in RASMCs. RASMCs were stimulated with AngII (1 μM) as the indicated times (A, C) and doses (B, D). Generation of ROS was measured in the presence of DCF-DA (20 μM). Fluorescence intensity was measured by MetaMorph software. Data are means ± S.D. of three independent experiments (n=3). Asterisks indicate statistical significance (p<0.05). Scale Bar, 200 μm.
- AngII-induced ROS generation is regulated by PI3K
Previously, it has been reported that PI3K is involved in the AngII signaling pathway. To examine whether PI3K is involved in the generation of ROS, kinase activity of PI3K was shunted by pharmacological inhibitor (LY294002, 10 μM). As shown in Fig. 2 , inhibition of PI3K completely blocked AngII-induced generation of ROS, whereas inhibition of ERK signaling pathway by PD98059 (10 μM) had no effect. These results indicate that PI3K plays an essential role in AngII-induced generation of ROS in RASMCs.
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PI3K signaling is required for ROS generation. (A) RASMCs were pretreated with various inhibitors such as PI3K inhibitor (LY294002, 10 μM), ERK inhibitor (PD98059, 10 μM) for 20 min. Fluorescent images were taken at 10× magnitude. (B) AngII-induced fluorescence intensity was measured by MetaMorph software. Data are means ± S.D. of three independent experiments (n=3). Asterisks indicate statistical significance (p<0.05). Scale Bar, 200 μm.
- AngII-induced calcium mobilization through PI3K and L-type calcium channel
Since activation of PI3K was necessary for AngII-induced generation of ROS ( Fig. 2 ), we examined whether PI3K activity is required for the AngII-induced calcium mobilization which plays an essential role in the production of ROS in VSMCs. As shown in Fig. 3 , inhibition of PI3K significantly attenuated AngII-induced calcium mobilization. However, inhibition of ERK did not affect. AngII-induced calcium mobilization was also significantly blocked by extracellular calcium chelation by EGTA. In addition, inhibition of L-type calcium channel by nifedipine (20 μM) significantly blocked AngII-induced calcium mobilization. These results indicate that AngII-induced calcium mobilization is mediated by PI3K and L-type calcium channel.
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PI3K and L-type calcium channel-dependent calcium mobilization. (A) RASMCs were pretreated with each pharmacological inhibitor of PI3K (LY294002, 10 μM), ERK (PD98059, 10 μM), or L-type calcium channel (Nifedipine, 20 μM) for 20 min. In some condition, AngII-induced calcium mobilization was measured in the presence of EGTA (5 mM) in buffer. The time of AngII stimulation is indicated by an arrow head. (B) Calcium mobilization was measured as described in Materials and Methods Data are means ± S.D. of three independent experiments. Asterisks indicate statistical significance (p<0.05).
- ROS is generated by L-type calcium channel
Since calcium mobilization of RASMCs was regulated by L-type calcium channel, we examined the role of L-type calcium channel during AngII-induced generation of ROS. As shown in Fig. 4 , chelation of extracellular calcium by EGTA (5 mM) or calcium-free buffer strongly inhibited AngII-induced ROS generation. In addition, blocking of L-type calcium channels by nifedipine (20 μM) completely abolished AngII-induced ROS generation. These results suggest that ROS is generated by calcium mobilization which is regulated by L-type calcium channel.
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Lager Image
AngII-induced ROS generation is mediated by L-type calcium channel. (A) RASMCs were pretreated with EGTA (5 mM), nifedipine (20 μM), or incubated in calcium-free media for 1 hr, and followed by stimulation with AngII for 15 min. Images were taken under fluorescent microscope at 10× magnitude. (B) Fluorescence intensity was measured by MetaMorph software. Data are means ± S.D. of three independent experiments (n=3). Asterisks indicate statistical significance (p<0.05). Scale Bar, 200 μm.
ROS play an important role as modulators of AngII signal transduction in VSMC [16] . In the present study, we provided significant role of calcium mobilization by PI3K/L-type calcium channel during AngII-induced ROS generation. Plethora of reports suggests that AngII mainly activats p38 MAPK, ERK1/2 and Akt signaling pathways. In addition, our data support the idea that PI3K plays an essential role in the generation of ROS although the mechanism underlying activation of PI3K is still unclear.
Calcium mobilization plays critical roles in many VSMC physiologies. There are two main pathways that lead to intracellular calcium mobilization. First, activation of PLC generates IP 3 which bind to calcium channel on the surface of endoplasmic reticulum. Second, subsequent activation of G protein releases Gβγ subunit which in turn binds to p110 catalytic subunit of PI3K. Activation of PI3K activates L-type calcium channel on the plasma membrane thereafter stimulates calcium uptake from extracellular fluid [12 , 21] . In correlation with this, our results showed that inhibition of PI3K completely blocked AngII-induced calcium mobilization ( Fig. 3 ). In addition, depletion of extracellular calcium or inhibition of L-type calcium channel significantly abolished AngII-induced calcium mobilization ( Fig. 4 ). It is also noteworthy that inhibition of MAPK signaling pathway had no effect on the mobilization of calcium ( Fig. 3 ), indicating that MAPK signaling pathway is not involved in the calcium mobilization. Therefore, PI3K/L-type calcium channel axis seems to be important pathways during AngII-induced calcium mobilization.
Several lines evidence support that calcium mobilization is critical for the ROS generation in VSMCs. For example, reciprocal regulation of calcium and ROS retains many VSMC physiologies [17] . It is noteworthy that our results showed that inhibition of L-type calcium channel or depletion of extracellular calcium abolished AngII-induced calcium mobilization ( Fig. 3 ). In addition, blocking of L-type calcium channel and chelation of extracellular calcium completely blocked AngII-induced ROS generation ( Fig. 4 ), suggesting that calcium mobilization is prerequisite for ROS generation. Particularly, our results demonstrated that calcium mobilization-dependent ROS generation was regulated by PI3K/L-type calcium channel pathways. Currently, we think that the source of ROS generation is NADPH oxidase since stimulation of VSMCs with nicotineamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitors such as diphenylene iodonium and apocynin (data shown). Currently, we are investigating the activation mechanism of NADPH oxidase by AngII stimulation.
Role of ROS seems to be diverse. AngII is involved in the pathogenesis of cardiac hypertrophy, hypertension and restenosis. For example, it has been observed that activation of NADPH oxidase and subsequent production of ROS is major leading cause of AngII-induced hypertension [13] . The molecular mechanism underlying ROS-dependent regulation of pathophysiologies are currently ambiguous, however, the function role of ROS seems to reside in the modulation of signaling cascade. For instance, AngII-dependent activation of Akt was attenuated by both NADPH oxdiase inhibitor (DPI) and overexpression of catalase, suggesting that ROS plays as a potential signal transducer linking AT 1 R to the PI3K/Akt pathway in VSMCs [6 , 18] . In addition to ROS-dependent activation of PI3K/Akt signaling pathway, activation of p38 MAPK and Jun-N-terminal kinase (JNK) requires AngII-dependent production of ROS [10 , 20] . More importantly, chronic production of ROS suppresses the expression of AT 1 R thereby counter balance the AngII signaling pathways [11] . Therefore, ROS plays role as a positive as well as negative regulator in AngII-mediated signaling pathways. In this regard, production of ROS in VSMCs may play key roles in AngII-dependent regulation VSMC physiology such as vasoconstriction, restenosis, and vascular hyperplasia.
In summary, AngII strongly induced the production of ROS in VSMCs. AngII-dependent activation of PI3K plays key role in the mobilization of intracellular calcium. PI3Kdependent calcium mobilization is mediated by L-type calcium channel. Finally, L-type calcium channel-dependent calcium mobilization is major responsible second messenger for the production of ROS in VSMCs. Therefore, it is likely that PI3K/L-type calcium channel signaling pathway seems to be major responsible pathways that regulate ROS generation in VSMCs.
This work was supported for two years by Pusan National University Research Grant.
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