Abstract
We hypothesized that, due to a cross-talk between cytoplasmic O2−-sources and intraluminally expressed xanthine oxidase (XO), intraluminal O2− is instrumental in mediating intraluminal (endothelial dysfunction) and cytosolic (p38 and ERK1/2 MAPKs phosphorylation) manifestations of vascular oxidative stress induced by endothelin-1 (ET-1) and angiotensin II (AT-II). Isolated guinea-pig hearts were subjected to 10-min agonist perfusion causing a burst of an intraluminal O2−. ET-1 antagonist, tezosentan, attenuated AT-II-mediated O2−, indicating its partial ET-1 mediation. ET-1 and Ang-T (AT-II + tezosentan) triggered intraluminal O2−, endothelial dysfunction, MAPKs and p47phox phosphorylation, and NADPH oxidase (Nox) and XO activation. These effects were: (i) prevented by blocking PKC (chelerythrine), Nox (apocynin), mitochondrial ATP-dependent K+ channel (5-HD), complex II (TTFA), and XO (allopurinol); (ii) mimicked by the activation of Nox (NADH); and mitochondria (diazoxide, 3-NPA) and (iii) the effects by NADH were prevented by 5-HD, TTFA and chelerythrine, and those by diazoxide and 3-NPA by apocynin and chelerythrine, suggesting that the agonists coactivate Nox and mitochondria, which further amplify their activity via PKC. The effects by ET-1, Ang-T, NADH, diazoxide, and 3-NPA were opposed by blocking intraluminal O2− (SOD) and XO, and were mimicked by XO activation (hypoxanthine). Apocynin, TTFA, chelerythrine, and SOD opposed the effects by hypoxanthine. In conclusion, oxidative stress by agonists involves cellular inside-out and outside-in signaling in which Nox-mitochondria-PKC system and XO mutually maintain their activities via the intraluminal O2−.
Highlights
Oxidative stress and endothelial dysfunction play a critical role in the pathogenesis of cardiovascular disease [1,2,3] and such states as post-ischemic inflammation and no-reflow phenomenon [4,5,6]
It is believed that cardiovascular risk factors, which function via such agents as angiotensin II (AT-II) and endothelin-1 (ET-1) [7,8,9], and ischemia/reperfusion acting via ET-1 [4,6,10] mediate the production of excess vascular reactive oxygen species (ROS), including superoxide (O2−)
Production, which would be similar to that induced by ischemia/reperfusion in our guinea pig heart model, a production that appeared to be mediated by ET-1 [10]
Summary
Oxidative stress and endothelial dysfunction play a critical role in the pathogenesis of cardiovascular disease [1,2,3] and such states as post-ischemic inflammation and no-reflow phenomenon [4,5,6]. The present study, which utilizes the isolated guinea-pig heart model [10], aimed to investigate the role of cross-talk between the vascular O2− sources in acute ET-1 and AT-II signaling to mediate O2− production and endothelial dysfunction, and MAPKs phosphorylation (i.e., intraluminal and cytosolic manifestations of the oxidative stress, respectively). Both agonists were studied because they may differentially activate MAPKs [24]. The detailed hypotheses tested in this study were those outlined in Scheme 1, and their rationales were as follows
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