Abstract-Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, nϭ6); aldosterone (0.75 g/H, SC, nϭ8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, nϭ8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, nϭ8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH 2 -terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosteroneinfused rats showed higher systolic blood pressure (165Ϯ5 mm Hg) and urinary excretion of protein (106Ϯ24 mg/d) than vehicle-infused rats (118Ϯ3 mm Hg and 10Ϯ3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23Ϯ0.02) than vehicle-infused rats (0.09Ϯ0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127Ϯ2 and 125Ϯ5 mm Hg), and the elevations of urinary excretion of protein (10Ϯ2 and 9Ϯ2 mg/day) or TBARS contents (0.08Ϯ0.01 and 0.11Ϯ0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.

beta-Adrenoceptor stimulation provokes cardiac oxidative stress. In the acute phase of ISO infusion, ROS are important activators of cardiac MAP kinase cascades; while, in the chronic phase, ROS may participate in cardiac remodeling, especially in respect to wall stiffness, based on fibrogenesis.

Abstract-Reactive oxygen species (ROS) participate in cardioprotection of ischemic reperfusion (I/R) injury via preconditioning mechanisms. Mitochondrial ROS have been shown to play a key role in this process. Angiotensin II (Ang II) exhibits pharmacological preconditioning; however, the involvement of NAD(P)H oxidase, known as an ROS-generating enzyme responsive to Ang II stimuli, in the preconditioning process remains unclear. We compared the effects of 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial ATP-sensitive potassium channels), apocynin (an NAD(P)H oxidase inhibitor), and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger) on pharmacological preconditioning by Ang II in rat cardiac I/R injury in vivo. Treatment with a pressor dose of Ang II before a 30-minute coronary occlusion reduced infarct size as determined 24 hours after reperfusion. The protective effects of Ang II were eliminated by pretreatment with 5-HD or apocynin, similar to tempol. Both 5-HD and apocynin suppressed the enhanced cardiac lipid peroxidation and activation of the apoptosis signal-regulating kinase/p38, c-Jun NH 2 -terminal kinase (JNK) pathways, but not the Raf/MEK/extracellular signal-regulated kinase pathway, elicited by acutely administered Ang II. Apocynin but not 5-HD suppressed Ang II-induced augmentations of the NAD(P)H oxidase complex formation (p47 phox , p22 phox , and Rac-1) and its activity in the heart. Finally, 5-HD suppressed superoxide production by isolated cardiac mitochondria without any effect on their respiration. These results suggest that the preconditioning effects of Ang II for cardiac I/R injury may be mediated by cardiac mitochondriaderived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation. Key Words: angiotensin Ⅲ antioxidants Ⅲ free radicals Ⅲ heart T he opening of cardiac mitochondrial ATP-sensitive potassium (mitoK ATP ) channels protects against subsequent ischemic reperfusion (I/R) tissue injury in the heart. 1,2 Receptor activation ligands, such as bradykinin, opioids, and acetylcholine, have been shown to trigger a preconditioning state 3 similar to mitoK ATP channel openers, which are inhibited by 5-hydroxydecanoate (5-HD), a specific mitoK ATP channel inhibitor. In the process of mitoK ATP channel-mediated preconditioning, the involvement of mitochondriaderived free radicals 4,5 and the posteffectors mitogen-activated protein (MAP) kinases, especially p38 MAP kinase 6 -8 has been strongly suggested. 1,9 Angiotensin II (Ang II) is well known as a powerful inducer of oxidative stress to cardiovascular tissues, and the reactive oxygen species (ROS) generated participate in Ang II-induced intracellular signaling pathways. 10 We demonstrated that acutely administered Ang II stimulates redoxsensitive cardiac MAP kinase activation, which was eliminated by tempol, a superoxide dismutase mimetic, in vivo. 11 To date, NAD(P)H oxidase has been considered a source of ROS corresponding to Ang II effects in c...

Abstract-Reactive oxygen species (ROS) are key mediators in signal transduction of angiotensin II (Ang II). However, roles of vascular mitochondria, a major intracellular ROS source, in response to Ang II stimuli have not been elucidated. This study aimed to examine the involvement of mitochondria-derived ROS in the signaling pathway and the vasoconstrictor mechanism of Ang II. Using 5-hydroxydecanoate (5-HD; a specific inhibitor of mitochondrial ATP-sensitive potassium [mitoK ATP ] channels) and tempol (a superoxide dismutase mimetic), the effects of Ang II and diazoxide (a mitoK ATP channel opener) were compared on redox-sensitive mitogen-activated protein (MAP) kinase activation in rat vascular smooth muscle cells (RVSMCs) in vitro and in rat aorta in vivo. Stimulation of RVSMCs by Ang II or diazoxide increased phosphorylated MAP kinases (ERK1/2, p38, and JNK), as well as superoxide production, which were then suppressed by 5-HD pretreatment in a dose-dependent manner, except for ERK1/2 activation by Ang II. The same events were reproduced in rat aorta in vivo. Ang II-like diazoxide depolarized the mitochondrial membrane potential (⌬⌿ M ) of RVSMCs determined by JC-1 fluorescence, which was inhibited by 5-HD. 5-HD did not modulate Ang II-induced calcium mobilization in RVSMCs and did not affect on the vasoconstrictor effect in either acute or chronic phases of Ang II-induced hypertension. These results reveal that Ang II stimulates mitochondrial ROS production through the opening of mitoK ATP channels in the vasculature-like diazoxide, leading to reduction of ⌬⌿ M and redox-sensitive activation of MAP kinase; however, generated ROS from mitochondria do not contribute to Ang II-induced vasoconstriction. Key Words: angiotensin Ⅲ oxidative stress R eactive oxygen species (ROS) are recognized as mediators of vascular signal transduction and are involved in activation of mitogen-activated protein (MAP) kinases. 1 Participation of ROS in blood pressure regulation has been shown in Ang II-induced hypertension 2-4 and other hypertensive models. [5][6][7] We recently demonstrated time-dependent transition of ROS sensitivity of Ang II hypertension in rats, in which in the early phase of Ang II infusion, high blood pressure does not depend on ROS production, but thereafter it shifts to being ROS-sensitive. 8 In this study, it was also found that in the acute phase of Ang II infusion, MAP kinases are stimulated through a ROS sensitive mechanism.Recent reports have supported the hypothesis that the enzyme NAD(P)H oxidase plays a major role as the most important source of superoxide anions in vascular cells and contributes significantly to the functional and structural alterations present in hypertension or atherosclerosis. 9,10 It has been further proposed that NAD(P)H oxidase is essential for production of superoxide in response to Ang II stimuli to vascular tissues. Mice deficient in the p47 phox gene showed significantly lower arterial blood pressure elevation during chronic Ang II infusion. 11 Moreover, endothelial ce...