Environmental information, survey date and time, and number of Swinhoe's Rails located during surveys at 186 sites.

The results of a survey of wintering Swinhoe's Rails Coturnicops exquisitus at 186 wet-grasslands in the Chugoku, Shikoku, and Kyushu regions of southwestern Japan, during the winters of 2021-2022 and 2022-2023. Survey sites studied in the daytime are indicated by *. </p

ARB induces eNOS and AMPK phosphorylation in HUVECs.

<p><b>A</b>. 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>B</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. <b>C</b>. 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>B</b>, <b>C</b>: *, p < 0.01 versus control; #, p < 0.01 versus Val; †, p < 0.05 versus Irbe; **, p < 0.01 versus Irbe. <b>D</b>. HUVECs were incubated with the vehicle (DMSO) or telmisartan (0.1, 1, and 10 µM) for 2 h. A representative blot is shown. <b>E</b>. Relative phosphorylation levels of eNOS were quantified as described in <b>B</b>. Bar graph shows the mean ± SEM of 4 independent experiments. <b>F</b>. Relative phosphorylation levels of AMPK were quantified as described in <b>C</b>. Bar graph shows the mean ± SEM of 4 independent experiments. <b>E</b>, <b>F</b>: *, 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. <b>G</b>. HUVECs were incubated with telmisartan (10 µM) for 15 min to 8 h. <b>H</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. <b>I</b>. 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. <b>H</b>, <b>I</b>: *, p < 0.05 versus control (0 min); #, p < 0.01 versus control.</p

AMPK inhibition does not inhibit eNOS phosphorylation induced by telmisartan in HUVECs.

<p><b>A</b>. 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>B</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.</p

Telmisartan-induced NO production is suppressed by p38 MAPK inhibition.

<p>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.</p

Telmisartan-induced eNOS phosphorylation levels after pretreatment with PI3K or PKA inhibitors.

<p><b>A</b>. 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>B</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. <b>C</b>. 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. <b>D</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. KT, KT5720. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus KT+DMSO; †, p < 0.05 versus H89+DMSO.</p

Immunohistochemical staining of aortic endothelium treated with valsartan and telmisartan.

<p><b>A</b>. 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>B</b>, <b>C</b>. 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.</p

Telmisartan-induced eNOS phosphorylation levels after pretreatment with PPARγ or PPARβ/δ inhibitors.

<p><b>A</b>. 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>B</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. <b>C</b>. 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. <b>D</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. GSK, GSK3787. *, p < 0.05 versus control (nc+DMSO); #, p < 0.05 versus GSK+DMSO.</p

Proposed scheme for endothelial eNOS and AMPK activation by telmisartan.

<p>Telmisartan activates eNOS and increases NO production via p38 MAPK, which is independent of AT1R.</p

AT1 inhibition does not inhibit eNOS or AMPK phosphorylation induced by telmisartan in HUVECs.

<p><b>A</b>. 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>B</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. <b>C</b>. 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.</p

Telmisartan induces p38 MAPK and CREB phosphorylation in HUVECs.

<p><b>A</b>. HUVECs were incubated with telmisartan for 15 min to 4 h to evaluate the phosphorylation levels of p38MAPK Thr180/Tyr182 and CREB Ser133. <b>B</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. <b>C</b>. 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>B</b>, <b>C</b>: *, p < 0.05 versus control (0 min); #, p < 0.01 versus control. <b>D</b>. 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. <b>E</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. SB, SB202190. *, p < 0.01 versus control (nc+DMSO). <b>F</b>. 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. <b>G</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