Upregulation of Orai Channels Contributes to Aging-Related Vascular Alterations in Rat Coronary Arteries

Abstract

Vascular territories display heterogeneous sensitivity to the impacts of aging. The relevance of the STIM/Orai system to vascular function depends on the vascular bed. We aimed to evaluate the contribution of the STIM/Orai system to aging-related vascular dysfunction in rat coronary circulation. Vascular function was evaluated according to myography in coronary arteries from young (three-month-old) and older (twenty-month-old) rats. The effects of aging and STIM/Orai inhibition on the contraction and relaxation of the coronary arteries and on the protein expression of STIM-1, Orai1, and Orai3 in these vessels were determined. Aging-related hypercontractility to serotonin and endothelin-1 in arteries from male rats was reversed by STIM/Orai inhibition with YM-58483 or by specifically blocking the Orai1 channel with Synta66. The inhibitory effects of Synta66 on coronary vasoconstriction were also observed in older female rats. YM-58483 relaxed serotonin- but not KCl-contracted arteries from males. STIM/Orai inhibition improved defective endothelial vasodilations in aged arteries, even in the presence of NO synthase and cyclooxygenase inhibitors, but not in KCl-contracted segments. YM-58483 significantly enhanced relaxations to calcium-activated potassium channel stimulation in aged vessels. Increased protein expression of Orai1 and Orai3 was detected in arterial homogenates and sections from older rats. Upregulation of the Orai channel contributes to aging-related coronary dysfunction, revealing a potential target in reducing CVD risk.

Keywords: Orai channel; arterial hypercontractility; coronary artery; endothelial function; vascular aging.

Figure 1

STIM/Orai inhibition reduces the aging-related enhancement of serotonin-induced contractions in rat coronary arteries. Panel (A) shows the effects of the treatment with the vehicle (0.2% DMSO) or the STIM/Orai inhibitor, YM-58483 (YM, 20 µM), on serotonin (5-HT, 1 nM to 100 µM)-induced contractions in coronary arteries from young (3-month-old, 3 M) and old (20-month-old, 20 M) rats. Panel (B) shows the effects of the vehicle (0.2% DMSO) or YM (20 µM) on 5-HT-induced contractions in coronary arteries from older rats where the endothelium was previously removed. The lower panels show the effects of the treatment with the vehicle (0.1% DMSO) or the specific Orai1 inhibitor, synta66 (10 µM), on 5-HT-induced contractions in coronary arteries from older male (C) and female (D) rats. Data are expressed as the mean± S.E.M. of the percentage of 125 mM K⁺-induced contraction. n indicates the number of animals used for the experiments. *** indicates p < 0.001 vs. 3 M, †† p < 0.01, ††† p < 0.001 vs. vehicle via two-factor ANOVA.

Figure 2

Aging-related enhancement of endothelin-1-induced contractions is reduced by STIM/Orai inhibition in rat coronary arteries. Panel (A) shows the effects of the treatment with the vehicle (0.2% DMSO) or the STIM/Orai inhibitor YM-58483 (YM, 20 µM) on endothelin-1 (ET-1, 0.1 nM to 0.3 µM)-induced contractions in coronary arteries from young (3-month-old, 3 M) and older (20-month-old, 20 M) rats. Panels (B,C) show the effects of the treatment with the vehicle (0.1% DMSO) and the specific Orai1 inhibitor, synta66 (10 µM), on ET-1-induced contractions in coronary arteries from young and older male rats (B) and from older female rats (C). Data are expressed as the mean ± S.E.M. of the percentage of 125 mM K⁺-induced contractions. n indicates the number of animals used for the experiments. * indicates p < 0.05, ** p < 0.01 vs. 3 M, ††† p < 0.001 vs. the vehicle according to two-factor ANOVA.

Figure 3

STIM/Orai inhibition caused the relaxation of serotonin-precontracted coronary arteries from male rats. Representative tracings of relaxations induced by the cumulative addition of YM-58483 (0.1 to 30 µM) in coronary arteries from young (3-month-old, 3 M) (A,C) and older (20-month-old, 20 M) (B,D) rats precontracted with serotonin (5 HT) (A,B) or KCl (K⁺) (C,D). PAP indicates papaverine (0.1 mM). Panel (E) shows the quantification of YM-58483-induced relaxations. Data are expressed as the mean ± S.E.M. of the percentage of maximum relaxation induced by papaverine at the end of the experiment. n indicates the number of animals used for the experiments. ** indicates p < 0.01 vs. 3 M, ††† p < 0.001 vs. 5 HT-contracted segments, assessed by two-factor ANOVA.

Figure 4

Aging-related impairment of endothelium-dependent vasodilation is alleviated by STIM/Orai inhibition in coronary arteries from male rats. The upper panels show the effects of the treatment with the vehicle (0.1% DMSO) or the STIM/Orai inhibitor YM-58483 (YM, 10 µM) on acetylcholine (ACh, 1 nM to 10 µM)-induced relaxation in coronary arteries from young (3-month-old, 3 M) and older rats (20-month-old, 20 M) contracted with serotonin (5 HT, 1–10 µM) under control conditions (A) or after treatment with the NO synthase inhibitor L-NAME (100 µM) and the cyclooxygenase inhibitor indomethacin (10 µM) (B), as well as in those contracted with KCl (K⁺, 50 mM) and treated with indomethacin (C). The lower panels show the effects of the treatment with the vehicle or YM on relaxations induced by the NO donor sodium nitroprusside (SNP, 1 nM to 10 µM) (D), and the SK_Ca and IK_Ca stimulator NS309 (0.1 to 30 µM) (E), in coronary arteries from 3 M and 20 M rats contracted with 5 HT. Data are expressed as the mean ± S.E.M. of the percentage of maximum relaxation induced by papaverine (0.1 mM) at the end of the experiment. n indicates the number of animals used for the experiments. ** indicates p < 0.01, *** p < 0.001 vs. 3 M, †† p < 0.01, ††† p < 0.001 vs. vehicle according to two-factor ANOVA.

Figure 5

Orai1 and Orai3 channels are upregulated in rat coronary arteries from older male rats. Panel (A) shows representative immunoblots for the detection of STIM-1, Orai1, and Orai3, and corresponding α-actin in coronary artery homogenates from young (3-month-old, 3 M) and older rats (20-month-old, 20 M). The right panels show the quantification of the expression assays for STIM-1 (B), Orai1 (C), and Orai3 (D). Data are expressed as the mean ± S.E.M. of STIM-1, Orai1, and Orai3 band intensities normalized by respective α-actin band intensities. n indicates the number of animals from which the tissues were collected for the experiments. * indicates p < 0.05 vs. 3 M according to the Mann–Whitney U test. The lower panels show representative immunofluorescence images for the detection (green fluorescence) of STIM-1 (E,H), Orai1 (F,I), and Orai3 (G,J) in cryosections of coronary arteries from young (3 M) (E–G) and older rats (20 M) (H–J). The increased immunodetection of Orai1 and Orai3 was noted in vascular preparations from older rats (arrows). Nuclei are counterstained in blue. Magnifications: ×200. Panels (K–M) show the quantification of immunofluorescence for STIM-1 (K), Orai1 (L), and Orai3 (M), confirming the upregulation of Orai1 and Orai3 proteins in coronary arteries from older rats. Data are expressed as the mean ± S.E.M. of the percentage of average fluorescence obtained in arteries from 3 M rats. N indicates the number of animals from which the sections were collected for the experiments. ** indicates p < 0.01 vs. 3 M according to the Mann–Whitney U test.