Figures
Abstract
Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.
Citation: Myojo M, Nagata D, Fujita D, Kiyosue A, Takahashi M, Satonaka H, et al. (2014) Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells. PLoS ONE 9(5): e96948. https://doi.org/10.1371/journal.pone.0096948
Editor: Ryuichi Morishita, Osaka University Graduate School of Medicine, Japan
Received: February 24, 2014; Accepted: April 13, 2014; Published: May 14, 2014
Copyright: © 2014 Myojo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by Grants-in-Aid #22590823 (to DN) and #17659229 (to Y.H.), Core Research for Evolutional Science and Technology (to Y.H.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Sankyo Foundation of Life Science (to D.N.), and by a Research Grant from the Fugaku Trust for Medical Research (to D.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The renin-angiotensin system (RAS) is one of the most pivotal mechanisms that modulate blood pressure. Furthermore, it is known to be the upstream cause of various cardiovascular diseases such as atherosclerosis, hypertension, ventricular hypertrophy, myocardial infarction, and heart failure [1]. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) are frequently prescribed for the treatment of hypertension. They also reduce the risk of renal dysfunction and the incidence of cardiovascular diseases [1], [2], [3], [4], [5]. The effects of RAS inhibitors are mostly common within the same drug class, but some effects are reported to be drug-specific. Among ARBs, the unique function of telmisartan in the adipose tissue is mediated through peroxisome proliferator-activated receptor (PPAR)γ activation. However, the mechanism of the vascular protective effect of telmisartan is not fully understood. Both endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK) have been suggested to play a role in the vascular endothelium to protect against the deteriorating effects of oxidative stress. Recently, post-translational phosphorylation of eNOS by kinases is considered to play an important role in regulation of eNOS activity [6], [7]. Five phosphorylation sites in eNOS have been identified and Ser1177 is considered to be the most important phosphorylation site of its enzyme activity. AMPK plays a protective role in vascular endothelial cells through cellular autophagy and by suppression of apoptosis [8]. It has also been reported that AMPK is essential for angiogenesis in response to hypoxic stress [9].
In this study, we examined the vascular protective effect of ARBs with relation to their ability to activate eNOS and AMPK. Additionally, we examined which signaling pathway plays a pivotal role in eNOS activation by telmisartan in human umbilical vein endothelial cells (HUVECs).
Materials and Methods
Materials
Antibodies against phospho-eNOS (Ser1177), phospho-AMPKα (Thr172), AMPKα, phospho-Akt (Ser473), phospho-p38 mitogen-activated protein kinase (p38 MAPK) (Thr180/Tyr182), p38 MAPK and cAMP response element binding protein (CREB) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phospho-CREB (Ser133) and eNOS (NOS3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phospho-acetyl CoA carboxylase (ACC) (Ser79) antibody was purchased from Millipore (Billerica, MA). An anti-myc tag antibody was purchased from Upstate Biotechnology (Lake Placid, NY) and an anti-HA tag antibody was purchased from Roche (Basel, Switzerland).
Wortmannin, LY294002, and KT5720 were purchased from Cayman Chemical Company (Ann Arbor, MI). H89 and GSK3787 were purchased from Tocris Bioscience (Bristol, UK). GW9662 was purchased from Wako Pure Chemical (Osaka, Japan). SB202190 was purchased from Calbiochem (Darmstadt, Germany).
Valsartan and irbesartan were purchased from Vijayasri Chemicals (Andhra Pradesh, India). Telmisartan was provided by Boehringer Ingelheim (Ingelheim, Germany).
Adenovirus
The replication-defective adenoviral vector expressing dominant-negative AMPK was identical to that used in a previous report [10]. This vector overexpresses the rat AMPK α2-subunit in which lysine45 has been mutated to arginine and is fused in-frame with the c-Myc epitope tag. An adenoviral vector expressing green fluorescent protein (GFP) was obtained from Qbiogene (Illkirch, France) and used to assess transduction efficiency. An adenoviral vector expressing dominant negative p38 MAPK fused with a HA epitope tag was a generous gift from Dr. Mitsuyama and this was used in a previous report [11].
Cell culture and adenovirus infection
HUVECs were purchased from Kurabo (Osaka, Japan) and cultured in HuMedia EG2 (Kurabo). In some experiments, HUVECs were transduced with the indicated replication-defective adenoviral vectors at a multiplicity of infection (MOI) of 50 for 1 day. The medium was then changed to HuMedia basic medium (EB2) with 0.2% FBS to reduce stimulation by serum mitogens. In all experiments, HUVECs were used at passage 7 or less.
Drug treatments
After incubation in EB2 containing 0.2% FBS for 1 day, the cells were treated with inhibitors, antagonists and ARBs diluted in dimethyl sulfoxide (DMSO).
Western blot analysis
Western blotting was carried out as described previously [12], [13]. An ECL-PLUS Western Blotting Detection kit (GE Healthcare, Piscataway, NJ) was used for detection. The density of the bands was quantified using the Scion Image program. Each experiment was repeated 3–4 times.
Immunohistochemical staining of mouse aortic endothelium
The animal care and use procedures were approved by the Animal Care Committee of the University of Tokyo (Permit Number: P09-015). Male C57 BL mice were purchased from Oriental Yeast (Tokyo, Japan) and fed standard laboratory chow (CLEA Japan, Tokyo, Japan). After drug treatment for 7 days, the mice were sacrificed to extract the descending aortas. The tissues were fixed in 4% paraformaldehyde, dehydrated, and then embedded in paraffin. The paraffin-embedded tissues were cut into cross sections (2 µm). The sections were deparaffinized and rehydrated for immunohistochemistry. Tissue sections were incubated with rabbit anti-phospho eNOS (Ser1177) or -eNOS antibodies diluted at 1:200, followed by a biotinylated anti-rabbit secondary antibody and then horseradish peroxidase-labeled streptavidin, according to the manufacturer's instructions (DAKO, Copenhagen, Denmark). Staining intensities were measured in 30 randomly selected fields of the endothelium and media by Scion Image program. The intensity ratio of the endothelium to media was then compared statistically.
cGMP assay
To evaluate nitric oxide (NO) output from HUVECs, the intracellular cGMP concentration was measured using a cGMP enzyme immunoassay system (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions and our previous reports [8], [13]. After incubation in low serum medium for 8 h, the cells were incubated in serum-free medium containing telmisartan or other reagents for 4 h. Each experiment was performed using 4 independent samples for 3 separate experiments.
Results
Telmisartan phosphorylates eNOS Ser1177 and AMPKα Thr172 in HUVECs
Western blot analyses were performed to compare the phosphorylation levels of eNOS Ser1177 and AMPKα Thr172 in HUVECs stimulated by three different ARBs, valsartan, irbesartan, and telmisartan, at 10 µM. Significant increases were observed in eNOS and AMPKα phosphorylation levels following treatment with telmisartan, but not the vehicle, valsartan, or irbesartan (Fig. 1A–C).
A. HUVECs were incubated with the vehicle (DMSO), valsartan (10 µM), irbesartan (10 µM), or telmisartan (10 µM) for 2 h. Representative data are shown. B. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. C. Relative phosphorylation levels of AMPK calculated as the ratio of phospho-AMPK to total AMPK. Bar graph shows the mean ± SEM of 4 independent experiments. Val, valsartan; Irbe, irbesartan; Telmi, telmisartan. B, C: *, p < 0.01 versus control; #, p < 0.01 versus Val; †, p < 0.05 versus Irbe; **, p < 0.01 versus Irbe. D. HUVECs were incubated with the vehicle (DMSO) or telmisartan (0.1, 1, and 10 µM) for 2 h. A representative blot is shown. E. Relative phosphorylation levels of eNOS were quantified as described in B. Bar graph shows the mean ± SEM of 4 independent experiments. F. Relative phosphorylation levels of AMPK were quantified as described in C. Bar graph shows the mean ± SEM of 4 independent experiments. E, F: *, p < 0.01 versus control (DMSO); #, p < 0.05 versus 0.1 µM telmisartan; †, p < 0.01 versus 0.1 µM telmisartan; **, p < 0.05 versus 1 µM telmisartan. G. HUVECs were incubated with telmisartan (10 µM) for 15 min to 8 h. H. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. I. Relative phosphorylation levels of AMPK calculated as the ratio of phospho-AMPK to total AMPK. Bar graph shows the mean ± SEM of 4 independent experiments. H, I: *, p < 0.05 versus control (0 min); #, p < 0.01 versus control.
We also examined concentration-dependent differences in eNOS and AMPK phosphorylation levels induced by telmisartan. After 24 h of incubation in low serum, HUVECs were incubated with DMSO, telmisartan (0.1, 1, or 10 µM) for 2 h followed by protein extraction. In telmisartan-treated cells, phosphorylation levels of AMPK Thr172 and eNOS Ser1177 increased in a concentration-dependent manner (Fig. 1D–F). In valsartan- and irbesartan-treated cells, we found no significant upregulation of phosphorylation levels (data not shown).
Next, we examined time-dependent increases in eNOS and AMPK phosphorylation by telmisartan. HUVECs were incubated with telmisartan for 15 min to 8 h followed by protein extraction for western blot analyses. The ratio of phospho-eNOS (Ser1177)/eNOS significantly increased at 1 h and reached the maximum at 2 h. Furthermore, the ratio of phospho-AMPKα (Thr172)/AMPKα significantly increased from 1 h and reached the maximum at 8 h (Fig. 1 G–I).
Oral administration of telmisartan increases eNOS phosphorylation in the mouse endothelium of the descending aorta
Next, we examined whether oral administration of telmisartan could increase eNOS phosphorylation levels in vivo, as was observed in cultured HUVECs. C57 BL6 mice were treated with methyl cellulose (vehicle), valsartan (5 mg⋅kg−1⋅day−1) or telmisartan (2.5 mg⋅kg−1⋅day−1) for 7 days. At 4 h after the last administration, the mice were sacrificed and the aorta was isolated. Immunohistochemical analysis showed similar staining intensities of anti-eNOS antibody in valsartan and telmisartan groups, but the staining intensity of an anti-phospho-eNOS (Ser1177) antibody in the telmisartan group was almost 1.5 times higher than that in the valsartan group (Fig. 2A–C).
A. Mouse descending aortas after oral administration with valsartan (5 mg⋅kg−1⋅day−1) or telmisartan (2.5 mg⋅kg−1⋅day−1) for 7 days were immunostained using anti-phospho eNOS (Ser1177) and anti-eNOS antibodies. B, C. Staining intensities were measured in 30 randomly selected field of endothelium (E) and media (M). The intensity ratios of the endothelium to media were compared statistically.
Pretreatment with high concentrations of valsartan does not inhibit eNOS and AMPK phosphorylation by telmisartan in HUVECs
To examine whether telmisartan phosphorylates eNOS and AMPK through binding to AT1R, HUVECs were incubated with telmisartan for 2 h under a condition in which the AT1R binding pocket was blocked by 30 min of pretreatment with high concentrations of valsartan (200 µM). The results of western blot analyses showed that the levels of eNOS and AMPK phosphorylation induced by telmisartan were independent of pretreatment with valsartan (Fig. 3A, B).
A. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h under the conditions, in which the AT1R binding pocket was blocked by pretreatment with high concentrations of valsartan (200 µM) for 30 min. Representative immunoblots of eNOS and AMPK are shown. B. Relative phosphorylation levels of eNOS were calculated as the band intensity ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. C. Relative phosphorylation levels of AMPK were calculated as the band intensity ratio of phospho-AMPK to total AMPK. Bar graph shows the mean ± SEM of 4 independent experiments. nc, negative control. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus Val+DMSO; †, p < 0.01 versus control; **, p < 0.01 versus Val+DMSO.
Inhibition of either phosphatidylinositol-3 kinase (PI3K) or protein kinase A (PKA) does not inhibit telmisartan-induced eNOS phosphorylation in HUVECs
We evaluated the involvement of PI3K-Akt in telmisartan-induced eNOS phosphorylation. Pretreatment with PI3K inhibitors wortmannin (1 µM) or LY294002 (50 µM) for 30 min did not suppress eNOS phosphorylation induced by telmisartan, suggesting that PI3K signaling is independent of such eNOS phosphorylation (Fig. 4A, B). We next evaluated the involvement of PKA in telmisartan-induced eNOS phosphorylation by pretreatment with PKA inhibitors KT5720 (5 µM) and H89 (10 µM). Neither inhibitor suppressed telmisartan-induced eNOS phosphorylation, suggesting that PKA is not involved in such phosphorylation (Fig. 4C, D).
A. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after pretreatment with PI3K inhibitors wortmannin (1 µM) or LY294002 (50 µM) for 30 min. B. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. Wort, wortmannin; LY, LY294002. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus Wort+DMSO; †, p < 0.05 versus LY+DMSO. C. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after pretreatment with PKA inhibitors KT5720 (5 µM) or H89 (10 µM) for 30 min. D. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. KT, KT5720. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus KT+DMSO; †, p < 0.05 versus H89+DMSO.
PPAR inhibition does not inhibit telmisartan-induced eNOS phosphorylation
We next evaluated the involvement of PPARγ and PPARβ/δ in telmisartan-induced eNOS phosphorylation. eNOS Ser1177 phosphorylation induced by telmisartan was unchanged by pretreatment with the PPARγ antagonist GW9662 (10 µM) (Fig. 5 A, B) or PPARβ/δ antagonist GSK 3787 (10 µM) for 30 min (Fig. 5C, D).
A. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after pretreatment with the PPARγ inhibitor GW9662 (10 µM) for 30 min. B. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. GW, GW9662. *, p < 0.01 versus control (nc+DMSO); #, p < 0.01 versus GW+DMSO. C. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after pretreatment with the PPARβ/δ inhibitor GSK3787 (10 µM) for 30 min. D. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. GSK, GSK3787. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus GSK+DMSO.
AMPK inhibition does not inhibit eNOS phosphorylation by telmisartan in HUVECs
Next, we evaluated the involvement of AMPK in telmisartan-induced eNOS phosphorylation. Overexpression of dominant-negative AMPK by an adenoviral vector did not suppress telmisartan-induced eNOS phosphorylation (Fig. 6A,B), suggesting that AMPK is not necessary for eNOS Ser1177 phosphorylation induced by telmisartan.
A. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after overexpression of dominant-negative AMPK (dnAMPK) or GFP by adenoviral vectors. Phosphorylation levels of ACC indicate the activity of AMPK. Myc-tag expression was checked to verify overexpression of dnAMPK. B. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. *, p < 0.05 versus control (GFP+DMSO); #, p < 0.05 versus dnAMPK+DMSO.
Involvement of p38 MAPK activation in telmisartan-induced eNOS phosphorylation in HUVECs
We evaluated the phosphorylation levels of p38 MAPK and its downstream target CREB in telmisartan-treated HUVECs. CREB Ser133 is a representative phosphorylation site of p38 MAPK. HUVECs were incubated with telmisartan for 15 min to 4 h followed by protein extraction. The band intensity ratios, phospho-CREB Ser133/CREB, and phospho-p38 MAPK Thr180/Tyr182/p38 MAPK, were significantly increased by telmisartan treatment and it reached a maximum at 2 h (Fig. 7A–C).
A. HUVECs were incubated with telmisartan for 15 min to 4 h to evaluate the phosphorylation levels of p38MAPK Thr180/Tyr182 and CREB Ser133. B. Relative phosphorylation levels of CREB calculated as the ratio of phospho-CREB (Ser133) to total CREB. Bar graph shows the mean ± SEM of 4 independent experiments. C. Relative phosphorylation levels of p38 MAPK calculated as the ratio of phospho-p38 MAPK to total p38 MAPK. Bar graph shows the mean ± SEM of 4 independent experiments. B, C: *, p < 0.05 versus control (0 min); #, p < 0.01 versus control. D. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h under the condition of p38 MAPK inhibition by pretreatment with SB202190 (10 µM) for 30 min. E. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. SB, SB202190. *, p < 0.01 versus control (nc+DMSO). F. HUVECs were incubated with the vehicle (DMSO) or telmisartan for 2 h after overexpression of dominant-negative p38 MAPK (dn p38 MAPK) or GFP by adenoviral vectors. HA-tag expression was checked to verify overexpression of dn p38 MAPK. G. Relative phosphorylation levels of eNOS calculated as the ratio of phospho-eNOS to total eNOS. Bar graph shows the mean ± SEM of 4 independent experiments. *, p < 0.05 versus control (GFP+DMSO).
We next evaluated the involvement of p38 MAPK in telmisartan-induced eNOS phosphorylation by using the p38 MAPK inhibitor SB202190 (10 µM). After SB202190 pretreatment, telmisartan-induced eNOS phosphorylation was suppressed to basal levels (Fig. 7D, E). We also investigated whether telmisartan-induced phosphorylation of AMPKα Thr172 could be suppressed by SB202190. However we did not find such inhibitory effects after the pre-incubation with the p38 MAPK inhibitor SB 202190 (data not shown).
Finally, we evaluated the specificity of p38 MAPK involvement in telmisartan-induced eNOS phosphorylation by using dominant negative p38 MAPK overexpression by a replication-defective adenoviral vector. Overexpression of dominant negative p38 MAPK significantly inhibited telmisartan-induced eNOS phosphorylation, suggesting that the p38 MAPK pathway is predominately involved in telmisartan-induced eNOS phosphorylation. (Fig. 7F, G).
Telmisartan-induced NO production is suppressed by p38 MAPK inhibition
To evaluate whether telmisartan-induced eNOS Ser1177 phosphorylation could increase NO output by activated p38 MAPK, we measured intracellular cGMP concentrations to estimate NO production by HUVECs treated with telmisartan. HUVECs were incubated with the vehicle (DMSO) or telmisartan (10 µM) for 2 h with or without 30 min of pretreatment with SB202190 (10 µM). Telmisartan increased the cGMP concentration by about 2 times compared with that in the control. However, after SB202190 pretreatment, telmisartan did not significantly increase the intracellular cGMP concentration in HUVECs (Fig. 8).
Intracellular cGMP concentrations were measured to evaluate NO output from HUVECs treated with telmisartan after corrected for the protein concentration. HUVECs were incubated with vehicle (DMSO) or telmisartan (10 µM) for 2 h with or without SB202190 (10 µM) pretreatment for 30 min. nc, negative control. *, p < 0.01 versus nc + DMSO; #, p < 0.01 versus nc + telmisartan.
Discussion
Telmisartan is reported to activate PPARγ, which is considered to ameliorate metabolic complications. PPARγ activation has been reported to inhibit vascular smooth muscle cell (VSMC) and fibroblast proliferation in vitro [14], [15]. Another study has shown that telmisartan is the only ARB that inhibits VSMC and cardiac fibroblast proliferation [16]. A clinical study evaluating the effects of telmisartan on the coronary plaque component and local inflammatory cytokines showed that telmisartan treatment leads to significant increases in fibrous volume and reductions in lipid volume on integrated backscatter intravascular ultrasound and significant decreases of matrix metalloproteinase (MMP)3, tumor necrosis factor-α, high-sensitive C-reactive protein and MMP9 sampled in coronary sinus [17].
In this study, we have focused on telmisartan-induced phosphorylation of eNOS and AMPK that are considered to be the dominant key players in vascular protection [18]. Telmisartan phosphorylated eNOS and AMPK significantly in HUVECs, but valsartan or irbesartan did not. Moreover, compared with valsartan, administration of telmisartan to mice significantly increased the phosphorylation of eNOS in the aortic endothelium.
We previously reported that AMPK not only protects vascular endothelium but also inhibits angiotensin II-induced VSMC proliferation [10]. Telmisartan-induced AMPK activation suggests that telmisartan has anti-arteriosclerotic functions via a different mechanism than that of ARBs in addition to the anti-arteriosclerotic functions through eNOS activation.
We investigated the intracellular signal transduction involved in telmisartan-induced eNOS phosphorylation. First, we examined the most important upstream kinase of eNOS, PI3K-Akt. Under various conditions such as pre- and post-conditioning in ischemia [19], [20], [21] and administration of insulin [22] and statins [23], [24], [25], [26], [27], [28], this pathway has been reported to play a pivotal role in cardioprotective and antiapoptotic effects. Two different kinds of PI3K inhibitor, wortmannin and LY294002, could not suppress telmisartan-induced eNOS phosphorylation, suggesting that PI3K is not necessary for this linkage. Because PKA inhibitors, KT5720 or H89, did not suppress telmisartan-induced eNOS phosphorylation, the PKA pathway was not involved either. Furthermore, we examined PPARγ and PPARβ/δ which are involved in the pleiotropic effects of telmisaratan [29], but telmisartan-induced eNOS phosphorylation was not suppressed by either GW9662 or GSK 3787, suggesting that both PPARγ and PPARβ/δ pathways were irrelevant to telmisartan-phospho-eNOS linkage.
Because AMPK has been reported to be one of the major upstream kinases of eNOS, we investigated the involvement of AMPK in telmisartan-induced eNOS Ser1177 phosphorylation by using dominant negative AMPK overexpression to inhibit the AMPK pathway completely. We observed that dominant negative AMPK overexpression did not inhibit telmisartan-induced eNOS phosphorylation completely, suggesting that AMPK was not responsible for this telmisartan-eNOS cascade. Moreover, we found that telmisartan increased phosphorylation levels of CREB Ser133 and p38 MAPK Thr180/Tyr182. Inhibition of p38 MAPK by SB202190 or overexpression of dominant negative p38 MAPK significantly suppressed telmisartan-induced eNOS phosphorylation. Furthermore, pretreatment with SB202190 significantly inhibited telmisartan-induced cGMP up-regulation in HUVECs. These results revealed that telmisartan increased eNOS phosphorylation and NO output via p38 MAPK (Fig. 9).
Telmisartan activates eNOS and increases NO production via p38 MAPK, which is independent of AT1R.
Some previous reports have shown the association of eNOS Ser1177 phosphorylation with the p38 MAPK pathway. Kan et al. reported that 17β-estradiol increases both the expression and Ser1177 phosphorylation levels of eNOS via p38 MAPK activation [30]. They suggested that this upregulation of eNOS activity leads to inhibition of ischemic myocardial damage in rats. Anter et al. reported that black tea polyphenols activate the p38 MAPK pathway, which increases eNOS activity via Ser1177 phosphorylation and Thr495 dephosphorylation via Akt [31]. Furthermore, Grossini et al. suggested that levosimendan leads to vasodilation through NO production via p38 MAPK, ERK, and Akt pathway activation in porcine coronary endothelium [32]. p38 MAPK is activated by stimulation via cytokines and physiochemical stresses such as ultraviolet irradiation, fever, and osmotic pressure. However, the mechanism underlying telmisartan-mediated activation of the p38 MAPK pathway remains to be resolved. Telmisartan is more lipid soluble than other ARBs, and this property might be one of the reasons for p38 MAPK activation in HUVECs. Because some anti-allergy drugs are reported to affect the function of membrane-bound proteins and induce signal transduction, including calcium pathways [33], telmisartan might induce structural changes of the cell membrane and transduce phosphorylation signals. Apoptosis signal regulating kinase-1(ASK1) is one of the most pivotal upper kinase of p38 MAPK. Although functions of this kinase have been reviewed in details [34], the precise mechanisms how this kinase is activated by extracellular stress or DNA damages have been unknown. We have to investigate in the future the mechanism of telmisartan-induced p38 MAPK activation, which might include ASK1 or other upper kinases activation.
In addition to its primary role to antagonize AT1 receptor, telmisartan has unique properties which might ameliorate metabolic diseases and vascular complications via different pathway from PPARγ. We expect that a variety of clinical benefits of telmisartan concerning its unique properties would be analyzed in details and fully uncovered in the near future.
Acknowledgments
We gratefully acknowledge the excellent technical support of Ms Asuka Ishii and Ms Marie Morita.
Author Contributions
Conceived and designed the experiments: DN. Performed the experiments: MM DF AK MT HS. Analyzed the data: MM. Wrote the paper: MM. Critically revised the manuscript: YM TA RN IK YH. Approved the final manuscript submitted: YH.
References
- 1. Ferrario CM, Strawn WB (2006) Role of the renin-angiotensin-aldosterone system and proinflammatory mediators in cardiovascular disease. Am J Cardiol 98: 121–128.
- 2. Pitt B (2002) Clinical trials of angiotensin receptor blockers in heart failure: what do we know and what will we learn? Am J Hypertens 15: 22S–27S.
- 3. Kjeldsen SE, Julius S (2004) Hypertension mega-trials with cardiovascular end points: effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. Am Heart J 148: 747–754.
- 4. Schmieder RE, Hilgers KF, Schlaich MP, Schmidt BM (2007) Renin-angiotensin system and cardiovascular risk. Lancet 369: 1208–1219.
- 5. Giles TD (2007) Renin-angiotensin system modulation for treatment and prevention of cardiovascular diseases: toward an optimal therapeutic strategy. Rev Cardiovasc Med 8 Suppl 2S14–S21.
- 6. Michel T, Li GK, Busconi L (1993) Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A 90: 6252–6256.
- 7. Corson MA, James NL, Latta SE, Nerem RM, Berk BC, et al. (1996) Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 79: 984–991.
- 8. Nagata D, Kiyosue A, Takahashi M, Satonaka H, Tanaka K, et al. (2009) A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell. Hypertens Res 32: 133–139.
- 9. Nagata D, Mogi M, Walsh K (2003) AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J Biol Chem 278: 31000–31006.
- 10. Nagata D, Takeda R, Sata M, Satonaka H, Suzuki E, et al. (2004) AMP-activated protein kinase inhibits angiotensin II-stimulated vascular smooth muscle cell proliferation. Circulation 110: 444–451.
- 11. Kawano H, Kim S, Ohta K, Nakao T, Miyazaki H, et al. (2003) Differential contribution of three mitogen-activated protein kinases to PDGF-BB-induced mesangial cell proliferation and gene expression. J Am Soc Nephrol 14: 584–592.
- 12. Nagata D, Hirata Y, Suzuki E, Kakoki M, Hayakawa H, et al. (1999) Hypoxia-induced adrenomedullin production in the kidney. Kidney Int 55: 1259–1267.
- 13. Nagata D, Takahashi M, Sawai K, Tagami T, Usui T, et al. (2006) Molecular mechanism of the inhibitory effect of aldosterone on endothelial NO synthase activity. Hypertension 48: 165–171.
- 14. Schupp M, Janke J, Clasen R, Unqer T, Kintscher U (2004) Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation 109: 2054–2057.
- 15. Benson SC, Pershadsingh HA, Ho CI, Chittiboyina A, Desai P, et al. (2004) Identification of telmisartan as a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity. Hypertension 43: 993–1002.
- 16. Benson SC, Iguchi R, Ho CI, Yamamoto K, Kurtz TW (2008) Inhibition of cardiovascular cell proliferation by angiotensin receptor blockers: are all molecules the same? J Hypertens 26: 973–980.
- 17. Yamaguchi K, Wakatsuki T, Soeki T, Niki T, Taketani Y, et al. (2014) Effects of telmisartan on inflammatory cytokines and coronary plaque component as assessed on integrated backscatter intravascular ultrasound in hypertensive patients. Circ J 78: 240–247.
- 18. Nagata D, Hirata Y (2010) The role of AMP-activated protein kinase in the cardiovascular system. Hypertens Res 33: 22–28.
- 19. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM (2004) Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 95: 230–232.
- 20. Zhang J, Baines CP, Zonq C, Cardwell EM, Wang G, et al. (2005) Functional proteomic analysis of a three-tier PKCepsilon-Akt-eNOS signaling module in cardiac protection. Am J Physiol Heart Circ Physiol 288: H954–H961.
- 21. Zhu M, Feng J, Lucchinetti E, Fischer G, Xu L, et al. (2006) Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. Cardiovasc Res 72: 152–162.
- 22. Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, et al. (2002) Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 105: 1497–1502.
- 23. Birnbaum Y, Ye Y, Rosanio S, Tavackoli S, Hu ZY, et al. (2005) Prostaglandins mediate the cardioprotective effects of atorvastatin against ischemia-reperfusion injury. Cardiovasc Res 65: 345–355.
- 24. Wolfrum S, Dendorfer A, Schutt M, Weidtmann B, Heep A, et al. (2004) Simvastatin acutely reduces myocardial reperfusion injury in vivo by activating the phosphatidylinositide 3-kinase/Akt pathway. J Cardiovasc Pharmacol 44: 348–355.
- 25. Yamakuchi M, Greer JJ, Cameron SJ, Matsushita K, Morrell CN, et al. (2005) HMG-CoA reductase inhibitors inhibit endothelial exocytosis and decrease myocardial infarct size. Circ Res 96: 1185–1192.
- 26. Bell RM, Yellon DM (2003) Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival pathway. J Am Coll Cardiol 41: 508–515.
- 27. Bell RM, Yellon DM (2003) Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol Cell Cardiol 35: 185–193.
- 28. Gonon AT, Bulhak A, Labruto F, Sjoquist PO, Pernow J (2007) Cardioprotection mediated by rosiglitazone, a peroxisome proliferator-activated receptor gamma ligand, in relation to nitric oxide. Basic Res Cardiol 102: 80–89.
- 29. He H, Yang D, Ma L, Luo Z, Ma S, et al. (2010) Telmisartan prevents weight gain and obesity through activation of peroxisome proliferator-activated receptor-delta-dependent pathways. Hypertension 55: 869–879.
- 30. Kan WH, Hsu JT, Ba ZF, Schwacha MG, Chen J, et al. (2008) p38 MAPK-dependent eNOS upregulation is critical for 17beta-estradiol-mediated cardioprotection following trauma-hemorrhage. Am J Physiol Heart Circ Physiol 294: H2627–H2636.
- 31. Anter E, Thomas SR, Schulz E, Shapira OM, Vita JA, et al. (2004) Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols. J Biol Chem 279: 46637–46643.
- 32. Grossini E, Molinari C, Caimmi PP, Uberti F, Vacca G (2009) Levosimendan induces NO production through p38 MAPK, ERK and Akt in porcine coronary endothelial cells: role for mitochondrial K(ATP) channel. Br J Pharmacol 156: 250–261.
- 33. Fischer MJ, Paulussen JJ, Roozendaal R, Tiemessen RC, de Mol NJ, et al. (1996) Relation between effects of a set of anti-allergic drugs on calcium pathways and membrane structure in Fc epsilon RI activated signal transduction. Inflamm Res 45: 564–573.
- 34. Matsuzawa A, Nishitoh H, Tobiume K, Takeda K, Ichijo H (2002) Physiological roles of ASK1-mediated signal transduction in oxidative stress- and endoplasmic reticulum stress-induced apoptosis: advanced findings from ASK1 knockout mice. Antioxid Redox Signal 4: 415–425.