Mitochondria-Derived Reactive Oxygen Species and Vascular MAP Kinases
Comparison of Angiotensin II and Diazoxide
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 [mitoKATP] channels) and tempol (a superoxide dismutase mimetic), the effects of Ang II and diazoxide (a mitoKATP 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 (ΔΨM) 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 mitoKATP channels in the vasculature-like diazoxide, leading to reduction of ΔΨM and redox-sensitive activation of MAP kinase; however, generated ROS from mitochondria do not contribute to Ang II–induced vasoconstriction.
Reactive 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 hypertension2–4 and other hypertensive models.5–7 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 p47phox gene showed significantly lower arterial blood pressure elevation during chronic Ang II infusion.11 Moreover, endothelial cells and vascular smooth muscle cells from p47phox-deficient mice had less responsiveness to Ang II stimuli in the production of ROS.12 These findings clearly indicate that superoxide production by Ang II is regulated by the NAD(P)H oxidase-dependent mechanism. However, these results do not take into consideration mitochondria, which provide another major ROS source.
Superoxide is produced in mitochondria by complexes I and III of the electron transport chain,13 and the rate of ROS synthesis can be modulated by mitochondrial inner membrane potential (ΔΨM).14 Mitochondrial ATP-sensitive potassium (mitoKATP) channel openers depolarize the ΔΨM and stimulate mitochondrial ROS production.15 To examine the implication of mitochondria-derived ROS in Ang II–induced vasoconstriction and the vascular signaling pathway leading to MAP kinase activation,16 we examined the effects of 5-hydroxydecanoate (5-HD; a mitoKATP channel inhibitor) on Ang II–induced vascular MAP (extracellular signal-regulated kinase [ERK]1/2, p38, and JNK) kinase activation, superoxide generation, and the ΔΨM as compared with diazoxide (a mitoKATP channel opener). We further examined the effects of 5-HD and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol; a membrane permeable radical scavenger)17,18 on acutely and chronically Ang II–induced hypertension in in vitro conscious rats.
Rat vascular smooth muscle cells (RVSMCs) prepared by the explant method from descending thoracic aortas of 4-week-old male Sprague–Dawley rats (CLEA; Osaka, Japan) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum. RVSMCs were authenticated using immunohistochemical staining for smooth muscle α-actin (Sigma). Cultures showing 95% staining for α-actin between passages 5 and 7 were used. Before the experiments, cells were starved for 48 hours.
Measurements of Superoxide Generation of RVSMCs
Superoxide generation of RVSMCs was measured by the lucigenin chemiluminescence method and also visualized by dihydroethidium (DHE) fluorescence images. Methods are detailed in the online data supplement (available online at http://www.hypertensionaha.org).
Visualization of Changes in ΔΨM of RVSMCs
Imaging study for ΔΨM of RVSMC by JC-1 fluorescence is detailed in the online data supplement.
Intracellular Calcium Measurements of RVSMCs
Measurement of intracellular calcium of RVSMCs is detailed in the online data supplement.
Measurements of Phosphorylation of ERK1/2, p38, and JNK MAP Kinases
Measurements of phosphorylated levels of MAP kinases (ERK1/2, p38, and JNK) in the aorta and cultured RVSMCs were conducted as previously described.19 As controls for protein loading and transfer to membrane, the total amounts of each MAP kinase in all groups were essentially constant, as determined by direct immunoblotting (data not shown).
Ten-week-old male Sprague–Dawley rats were used. Preparation of acutely and chronically Ang II–infused rats was described previously, and all hemodynamic measurements were performed on conscious rats.4,19 In acute experiments, Ang II and diazoxide were given intravenously at a rate of 200 and 10 ng · kg−1 per minute, respectively. Tempol (Sigma) was given at a priming dose of 30 mg · kg−1, followed by infusion at a rate of 0.5 mg · kg−1 per minute. 5-HD (Sigma) was given at a dose of 10 mg · kg−1 5 minutes before the start of Ang II or diazoxide infusion. Arterial blood pressure was monitored continuously, and after 30 minutes rats were euthanized and the aorta taken, quickly frozen in liquid nitrogen (LN2), and then stored at −80°C. The effects of 5-HD and tempol on chronically Ang II–infused rats (for 14 days, 200 ng · kg−1 per minute subcutaneous; by Alzet osmotic mini-pump) were examined similarly to the acute experiments. Measurements of lipid peroxidation levels in plasma were described previously. All the surgical and experimental procedures were performed according to the guidelines for the care and use of animals as established by Kagawa University.
Values are expressed as the mean±SEM. Statistical significance between >2 groups was tested using 2-way ANOVA followed by Newman–Keuls test or unpaired 2-tailed Student t test as appropriate, and values of P<0.05 were considered to indicate statistical significance.
5-HD and Tempol Suppress MAP Kinase Activation by Ang II or Diazoxide in RVSMCs
As shown in Figure 1A,a 30-minute exposure to 100 nmol/L Ang II increased phosphorylation of ERK1/2, p38, and JNK MAP kinases in RVSMCs. Among these, Ang II–induced augmentation of phosphorylated p38 and JNK MAP kinases was suppressed by 5-HD dose dependently. However, 5-HD suppressed that of ERK1/2 with less intensity, and only the highest dose of 5-HD showed significance. Similarly to 5-HD, tempol suppressed Ang II–induced augmentation of phosphorylated p38 and JNK MAP kinases, and that of phosphorylated ERK1/2 kinase less effectively. As shown in Figure 1B,a 30-minute exposure to 200 μmol/L diazoxide augmented phosphorylation of all MAP kinases in RVSMCs, and the magnitude of induction by this dose of diazoxide was similar to 100 nmol/L Ang II. It is of note that both 5-HD and tempol suppressed all MAP kinase phosphorylation augmented by diazoxide more effectively than that of Ang II. Treatment with 5-HD or tempol at the highest dose alone had no effect on basal MAP kinase activities in RVSMCs (data not shown).
5-HD Suppresses Stimulated Superoxide Generation by Ang II or Diazoxide in RVSMCs
To gain insights into the relation between mitochondrial ROS and vascular MAP kinase activation, we studied the effects of 5-HD on superoxide generation using RVSMCs. As shown in Figure 2A, Ang II and diazoxide increased superoxide generation of RVSMCs during the incubation time determined by the lucigenin chemiluminescence method with a lucigenin concentration of 10 μmol/L. The enhanced superoxide generation by Ang II or diazoxide was suppressed by pretreatment with 5-HD in a dose-dependent manner. DHE fluorescent images of RVSMCs also support 5-HD–sensitive superoxide generation by Ang II or diazoxide in RVSMCs (Figure 2B).
Ang II and Diazoxide Depolarize ΔΨM in RVSMCs
To address the possibility that increases of phosphorylated MAP kinase and superoxide generation by Ang II and diazoxide are related to mitochondrial functions, the changes in ΔΨM of living RVSMCs by exposure to Ang II or diazoxide were visualized using dual simultaneous detection confocal microscopy (Figure 3). Before stimulation, a single cell included both green (JC-1 monomer) and red (J-aggregate) mitochondria, indicating that they were composed of a wide range of ΔΨM for different mitochondria.20 The number of red mitochondria decreased after the exposure to 300 nmol/L Ang II and 200 μmol/L diazoxide. These changes were blunted by pretreatment with 5-HD, indicating that the depolarization of ΔΨM by Ang II stimuli was mediated through the opening of mitoKATP channels.
Effects of 5-HD on Intracellular Calcium Mobilization by Ang II in RVSMCs
The interaction between mitochondrial and calcium mobilization is an important issue under some pathological circumstance, such as hypoxia.21 We examined the effect of 5-HD on Ang II–induced calcium mobilization in RVSMCs (Figure 4). Cultured RVSMCs revealed 75±8 nmol/L as a baseline of [Ca2+]i; 100 nmol/L Ang II induced a rapid increase of [Ca2+]i to 450±54 nmol/L, which was followed by a slow return toward baseline. Pretreatment with 5-HD (30 and 300 nmol/L), added 5 minutes before Ang II stimuli, had no significant effect on baseline or Ang II–induced rapid increase of [Ca2+]i.
5-HD and Tempol Suppress Vascular MAP Kinase Activation by Ang II and Diazoxide in Conscious Rats
Using catheterized conscious rats, the effects of 5-HD and tempol on aortic MAP kinase activation by acutely administrated Ang II or diazoxide were examined. As shown in the Table, arterial blood pressure was increased by 50 mm Hg soon after intravenous infusion of Ang II (200 ng · kg−1 per minute) and was decreased by 15 mm Hg during diazoxide infusion (10 ng · kg−1 per minute). Pretreatments of 5-HD (10 mg · kg−1) and tempol (30 mg · kg−1) did not modify any hemodynamic changes elicited by Ang II or diazoxide.
A 30-minute infusion of Ang II or diazoxide increased phosphorylation of aortic ERK1/2, p38, and JNK MAP kinases. As shown in Figure 5, augmentation of p38 and JNK MAP kinase phosphorylation by Ang II in the aorta was significantly suppressed by 5-HD, whereas that of ERK1/2 was not affected by 5-HD treatment. The augmented phosphorylation of these MAP kinases by diazoxide was completely normalized by treatment with 5-HD. Tempol effectively suppressed Ang II–induced and diazoxide-induced aortic MAP kinase activation.
5-HD Does Not Affect Blood Pressure of Chronically Ang II-Induced Hypertension
Previously, we demonstrated that hypertension of chronically Ang II–infused hypertensive rats is ROS dependent and that it can be normalized by tempol administration.8 To assess whether mitochondrial ROS is involved in the maintenance of chronic phase of Ang II hypertension, the effects of 5-HD were tested in chronically Ang II–infused hypertensive rats in which high arterial blood pressure (153±5 mm Hg) and elevated levels of plasma lipid peroxidation developed. As shown in Figure 6, 5-HD had no significant effects on arterial blood pressure and elevated levels of plasma lipid peroxidation in chronically Ang II–infused rats, whereas tempol successfully normalized blood pressure and plasma lipid peroxidation in this model. We also tested the effects of 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron), a membrane-permeable radical scavenger. The results showed a similar pattern to tempol treatment, as is shown in the online data supplement.
The significant finding of the present study is that 5-HD, a specific inhibitor of mitoKATP channels, suppressed Ang II–induced redox-sensitive vascular MAP kinase activation, in particular, p38 and JNK MAP kinases in rat aorta in vivo and in cultured RVSMCs in vitro. These observations resemble the results of diazoxide treatment under both in vivo and in vitro conditions, indicating that acutely administered Ang II may activate mitoKATP channels and contribute to superoxide production in the vascular tissue. In support of this finding, we clearly demonstrated that Ang II reduced the ΔΨM of RVSMCs through the opening of mitoKATP channels in a similar manner to diazoxide.
We compared 2 stimulants, Ang II and diazoxide, because the latter is a mitochondrial-related compound and has been implicated in redox-sensitive phosphorylation and activation of ERK.22 Although Ang II and diazoxide have opposite actions for vascular tone, ie, as a vasoconstrictor and a vasodilator, both can activate aortic MAP kinases. The action of diazoxide is nonspecific to mitoKATP channels, and it also activates the sarcolemmal KATP channel. In our in vivo experiments, diazoxide decreased arterial blood pressure, which was caused by the opening of sarcolemmal KATP channels. However, the increases of MAP kinase activation by diazoxide may be caused by its action on mitoKATP but not on sarcolemmal KATP channels, because the vasodilator effect of diazoxide was not affected by pretreatment with 5-HD, a specific inhibitor of mitoKATP channels. The opening of mitoKATP channels by diazoxide increases superoxide generation in the heart.23 In this study, we confirmed that diazoxide did increase superoxide generation in RVSMCs, and that 5-HD suppressed the enhancing signals of superoxide by diazoxide, determined by lucigenin chemiluminescence and DHE fluorescence. In a related study, we found that 5-HD did not possess any radical scavenging activity (not shown), nor did it affect on activation of vascular NAD(P)H oxidase, a well-known target of Ang II (see the online data supplement). Thus, it seems likely that vascular mitoKATP channels are responsible for the production of superoxide by diazoxide and Ang II in vasculature, at least during the early phases of their actions. There have been few reports concerning the effects of Ang II on mitochondrial function; however, in this study we found a decrease in ΔΨM and augmented superoxide generation in response to Ang II stimuli of RVSMCs in a manner similar to that of diazoxide, supporting the hypothesis that the intracellular signaling of Ang II passes through mitochondrial functions, reaching redox-sensitive MAP kinase activation.1
In regard to the sensitivity of MAP kinase activation to mitochondrial ROS, a difference was seen in the case of ERK1/2 as activated by either Ang II or diazoxide. Previously, we have shown that the increase of phosphorylated MAP kinases in rat aorta by Ang II was eliminated by tempol.19 In this study, diazoxide-induced activation of ERK, p38, and JNK MAP kinases was also tempol-quenchable in the in vivo experiment, indicating a critical role of ROS for the activation of these 3 MAP kinases in vasculature. Furthermore, we found that 5-HD suppressed the increase of all the phosphorylated MAP kinases by diazoxide, and those of p38 and JNK MAP kinases by Ang II, but not that of ERK1/2 by Ang II. Although we used a high dose of tempol to suppress Ang II–induced ERK1/2 activation in this study, the resistance of ERK1/2 to tempol treatment was also observed in the in vivo study.19 Taken together, these results suggest that mitochondrial-derived ROS could be responsible for the Ang II–induced stimuli of all MAP kinases, including ERK1/2. However, another pathway independent of mitochondrial ROS might be involved in the redox-sensitive activation of vascular ERK1/2.
Inappropriate production of ROS in vasculature contributes to the maintenance of hypertension. To assess the possibility that Ang II–induced enhancement of mitochondrial ROS generation contributes to Ang II–mediated vasoconstriction, we examined the effects of 5-HD on blood pressure of acutely and chronically Ang II–infused rats. We previously demonstrated that tempol normalizes blood pressure of chronically Ang II–infused hypertensive rats, but this is not the case for acute vasoconstrictor effect of Ang II.8 However, the data from this study clearly demonstrated that 5-HD does not affect on vasoconstriction in either acutely or chronically administered Ang II, indicating that mitochondrial ROS do not participate in the vasoconstrictor mechanisms of Ang II–induced hypertension. Because a rapid increase of intracellular calcium is a determinant of vasoconstrictor effects of Ang II,24 we also tested the effects of 5-HD on intracellular calcium induction of RVSMCs by Ang II. To support our in vivo observation, we confirmed that the calcium mobilization in RVSMCs by Ang II was not affected by mitoKATP channel inhibition. Torrecillas et al suggested the contribution of H2O2 in vasoconstrictor mechanisms of Ang II by showing that catalase prevented Ang II–induced myosin light chain phosphorylation and intracellular calcium mobilization of RVSMCs, and also inhibited Ang II–induced constriction of rat aortic rings.25 Waypa et al also have shown mitochondria-mediated calcium increases in hypoxia in pulmonary arterial myocytes through ROS generation from the electron transport chain.21 However, our in vivo and in vitro data arising from this study indicated that mitochondria-derived ROS could not influence contractility of vasculature during the chronic phase of Ang II–induced hypertension.
In conclusion, we studied the involvement of mitochondria-derived ROS in intracellular signal transduction and vasoconstriction by Ang II stimuli in vivo and in vitro. Our results reveal that Ang II stimulates mitochondrial ROS generation through the opening of mitoKATP channels in the vasculature, as does diazoxide, leading to the reduction of ΔΨM and redox-sensitive activation of MAP kinases, especially p38 and JNK, and that ROS generated from mitochondria do not contribute to Ang II–induced vasoconstriction.
The findings of the current study indicate an important association between Ang II and mitochondrial function in ROS generation of vasculature. Inhibition of the renin-angiotensin system attenuated age-associated changes in mitochondrial function.26 Mitochondria are a major subcellular source of ROS, which would promote oxidative damage to cell structures and functions.27 In this respect, studies using materials such as mitochondrial-deficient (ρ°) cells or cells separated from aged population should provide further understanding of Ang II physiology and assessments of therapeutic intervention with renin-angiotensin system inhibitors. In this study, we have not examined inhibitors of mitochondrial respiratory chains to identify the site of superoxide generation. It has been shown that rotenone, an inhibitor of complex I, had no effect on Ang II–induced p38 and JNK activation in human vascular smooth muscle cells.28 Further studies will be necessary to clarify the site of superoxide generation for redox-sensitive MAP kinase activation in vasculature.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan.
- Received October 13, 2004.
- Revision received October 31, 2004.
- Accepted January 14, 2005.
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