Matlubur Rahman

Role of NAD(P)H Oxidase- and Mitochondria-Derived Reactive Oxygen Species in Cardioprotection of Ischemic Reperfusion Injury by Angiotensin II

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 NH2-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 (p47phox, p22phox, 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 mitochondria-derived ROS enhanced through NAD(P)H oxidase via JNK and p38 mitogen-activated protein kinase activation.

Aldosterone Stimulates Reactive Oxygen Species Production through Activation of NADPH Oxidase in Rat Mesangial Cells

It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O(2)(-)) in intact cells. The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 +/- 18 to 346 +/- 30 cpm/5 x 10(4) cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 micromol/L), significantly attenuated aldosterone-induced O(2)(-) production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.

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 (&Dgr;&PSgr;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 mitoKATP channels in the vasculature-like diazoxide, leading to reduction of &Dgr;&PSgr;M and redox-sensitive activation of MAP kinase; however, generated ROS from mitochondria do not contribute to Ang II–induced vasoconstriction.

Systemic and Regional Hemodynamic Responses to Tempol in Angiotensin II–Infused Hypertensive Rats

Recent studies have indicated that angiotensin II (Ang II) can stimulate oxidative stress. The present study was conducted to assess the contribution of oxygen radicals to hypertension and regional circulation during Ang II–induced hypertension. With radioactive microspheres, the responses of systemic and regional hemodynamics to the membrane-permeable, metal-independent superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) were assessed in conscious Ang II–infused hypertensive rats. Ang II–infused rats (80 ng/min SC for 12 days: n=25) showed higher mean arterial pressure (MAP: 161±4 mm Hg) and total peripheral resistance (TPR: 1.59±0.08 mm Hg · min−1 · mL−1) than vehicle-infused normotensive rats (116±3 mm Hg and 0.95±0.04 mm Hg · min−1 · mL−1, respectively; n=23). The blood flow rates in the brain, spleen, large intestine, and skin were significantly reduced in Ang II–infused rats compared with vehicle-infused rats, whereas rates in the lung, heart, liver, kidney, stomach, small intestine, mesenterium, skeletal muscle, and testis were similar. Vascular resistance was significantly increased in every organ studied except the lung, in which the resistance was similar. Tempol (216 &mgr;mol/kg IV) significantly reduced MAP by 30±4% from 158±7 to 114±5 mm Hg and TPR by 35±6% from 1.57±0.17 to 0.95±0.04 mm Hg · min−1 · g−1 in Ang II–infused rats (n=9) but had no effect on these parameters in vehicle-infused rats (n=8). In Ang II–infused rats, tempol did not affect regional blood flow but significantly decreased vascular resistance in the brain (29±6%), heart (31±6%), liver (37±7%), kidney (30±7%), small intestine (38±6%), and large intestine (47±7%). Ang II–infused hypertensive rats showed doubled vascular superoxide production (assessed with lucigenin chemiluminescence), which was normalized by treatment with tempol (3 mmol/L, n=7). Further studies showed that the NO synthase inhibitor, N&ohgr;-nitro-l-arginine methyl ester (11 &mgr;mol · kg−1 · min−1 IV, n=11) markedly attenuated the systemic and regional hemodynamic responses of tempol in Ang II–infused rats. These results suggest that in this model of hypertension, oxidative stress may have contributed to the alterations in systemic blood pressure and regional vascular resistance through inactivation of NO.

Aldosterone Stimulates Collagen Gene Expression and Synthesis Via Activation of ERK1/2 in Rat Renal Fibroblasts

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti–phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [3H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [3H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 &mgr;mol/L), a selective MR antagonist, or PD98059 (10 &mgr;mol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [3H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.

Role of Angiotensin II and Reactive Oxygen Species in Cyclosporine A-Dependent Hypertension

Abstract—Treatment with cyclosporine A (CysA), a potent immunosuppressive agent, is associated with systemic and renal vasoconstriction, leading to hypertension. The present study was conducted to elucidate the contribution of angiotensin II (Ang II) to CysA-induced hypertension and reactive oxygen species (ROS) generation. CysA (30 mg/kg per day SC), given for 3 weeks in rats, increased systolic blood pressure (SBP) from 119±2 to 145±3 mm Hg (n=7). Plasma and kidney Ang II levels were significantly higher in CysA-treated rats (136±10 fmol/mL and 516±70 fmol/g) than in vehicle-treated (1 mL olive oil) rats (76±10 fmol/mL and 222±21 fmol/g, n=7). CysA treatment increased AT1 receptor protein expression in the aorta (by 251±35%), whereas it was reduced in the kidney (by −32±4%). Superoxide anion production in aortic segments and kidney thiobarbituric acid–reactive substance (TBARS) contents were higher in CysA-treated rats (26±2 counts/min per milligram and 37±3 nmol/g) than in vehicle-treated rats (17±1 counts/min per milligram and 24±3 nmol/g). Concurrent administration of an AT1 receptor antagonist, valsartan (30 mg/kg per day, in drinking water), to CysA-treated rats (n=7) significantly decreased SBP (113±4 mm Hg) and prevented increases in vascular superoxide (16±2 counts/min per milligram) and kidney TBARS contents (21±3 nmol/g). Similarly, treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6,-tetramethylpiperidine-N-oxyl (Tempol; 3 mmol/L in drinking water, n=7), prevented CysA-induced increases in SBP (115±3 mm Hg), vascular superoxide (16±1 counts/min per milligram), and kidney TBARS contents (19±2 nmol/g). These data suggest that ROS generation induced by augmented Ang II levels contributes to the development of CysA-induced hypertension.

ROS During the Acute Phase of Ang II Hypertension Participates in Cardiovascular MAPK Activation But Not Vasoconstriction

Abstract—The relations among hypertensive response, oxidative stress, and mitogen-activated protein kinase (MAPK) in cardiovascular tissues have not been fully established. We investigated the involvement of reactive oxygen species on changes in the hemodynamics and cardiovascular MAPKs activities induced by acutely administered angiotensin II (Ang II) in conscious normotensive rats with or without treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol). Intravenous infusion of a pressor dose of Ang II rapidly increased mean arterial blood pressure (MBP) by 53±5 mm Hg. After a 30-minute treatment with Ang II, phosphorylated MAPKs (ERK1/2, JNK, p38) as well as thiobarbital reactive substances (T-BARS) were increased in the aorta and cardiac left ventricle. Tempol had no significant effect on the elevation of MBP elicited by Ang II; however, it dose-dependently suppressed the augmented phosphorylation of cardiovascular MAPKs and increased T-BARS levels in plasma and tissues induced by Ang II. An acutely administered pressor dose of phenylephrine, an &agr;-adrenoceptor agonist, also showed tempol-sensitive cardiovascular MAPK activation and tempol-insensitive blood pressure elevation. These in vivo data indicate that acute administration of Ang II or phenylephrine provoked an increase in oxidative stress in the cardiovascular tissues leading to the activation of MAPKs, whether it was mediated by pressure overload or the direct action of these vasoconstrictors, and that oxidative stress might not have a major contribution to the acute hypertensive responses elicited by the vasoconstrictors.

Contribution of reactive oxygen species to the pathogenesis of left ventricular failure in Dahl salt-sensitive hypertensive rats: effects of angiotensin II blockade.

Objective We investigated the contribution of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) generation to the pathogenesis of diastolic heart failure (DHF) in Dahl salt-sensitive (DS) hypertensive rats, with the aim of testing our hypothesis that the cardioprotective effects of angiotensin II (Ang II) blockade are provided by the suppression of this pathway. Methods DS rats were maintained on high (H: 8.0% NaCl) or low (L: 0.3% NaCl) salt diets from age 7 to 17 weeks. DS/H rats were also treated with candesartan cilexetil (10 mg/kg per day, orally) or a superoxide dismutase mimetic, tempol (3 mmol/l in drinking water) from age 7 to 17 weeks. Results DS/H rats represented hypertension, left ventricular (LV) relaxation abnormality and myocardial stiffening with preserved systolic heart function. As compared with DS/L rats, DS/H rats showed higher levels of transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), p22phox and gp91phox mRNA expression, NADPH oxidase activity and thiobarbituric acid-reactive substance (TBARS) contents in LV tissues. Gene expression of uncoupling protein-2 (UCP-2), an inner mitochondrial membrane proton transporter, was also 2.8 ± 0.5-fold higher. In DS/H rats, treatment with candesartan did not alter blood pressure, but resulted in a marked improvement of the hemodynamic deterioration; these therapeutic effects were accompanied by decreases in myocardial NADPH oxidase activity, TBARS contents and the expression of TGF-β, CTGF, p22phox, gp91phox and UCP-2. Similar therapeutic effects were provided by treatment with tempol in DS/H rats. Conclusions Our data suggest that NADPH oxidase-mediated ROS production contributes to the pathogenesis of DHF in DS hypertensive rats, and that the cardioprotective effects of AngII blockade are, at least partially, mediated through the suppression of this pathway.

Synergistic effect of mechanical stretch and angiotensin II on superoxide production via NADPH oxidase in vascular smooth muscle cells

Objective Mechanical forces and angiotensin II influence the structure and function of vascular cells, and play an important role in reactive oxygen species production. In this study, we examined the effects of mechanical stretch and angiotensin II on the expression of p22-phox and Nox-1, essential membrane components of NADPH oxidase, and superoxide production in rat vascular smooth muscle cells (VSMCs). Methods and results Neither a stretch force nor angiotensin II alone altered p22-phox and Nox-1 expression in VSMCs. Combined stimulation markedly increased p22-phox and Nox-1 mRNA, however, which was associated with increased NADPH oxidase activity, superoxide production and total 8-iso-prostaglandin F2α concentration. The increases in p22-phox mRNA levels induced by a stretch force in combination with angiotensin II were prevented by treatment with an angiotensin type I (AT1) receptor antagonist, RNH-6270 (100 nmol/l). Protein expression of the AT1 receptor was upregulated by a stretch force. Conclusions These data indicate that mechanical stretch and angiotensin II synergistically increase NADPH oxidase expression in VSMCs, and suggest that part of this mechanism is mediated through an upregulation of the AT1 receptor induced by mechanical stretch. The combined effects of mechanical strain and angiotensin II might promote vascular damage through the production of superoxide in a hypertensive state.

Effects of local administrations of tempol and diethyldithio-carbamic on peripheral nerve activity.

We have recently shown that systemic administration of a superoxide dismutase mimetic, tempol, resulted in decreases in mean arterial pressure and heart rate along with a reduction in renal sympathetic nerve activity (RSNA). It has also been shown that these parameters are significantly increased by systemic administration of a superoxide dismutase inhibitor, diethyldithio-carbamic (DETC), indicating a potential role of reactive oxygen species in the regulation of RSNA. In this study, we examined the effects of local administrations of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) and DETC on RSNA in anesthetized rats. Either tempol or DETC was directly administered onto the renal sympathetic nerves located between the electrode and ganglion. Local application of tempol (10 μL, 0.17 to 1.7 mol/L, n=6) resulted in dose-dependent decreases in integrated RSNA (by −81±6% at 1.7 mol/L) without alterations in mean arterial pressure and heart rate. In contrast, DETC (10 μL, 0.17 to 1.7 mol/L, n=6) increased RSNA dose-dependently. The responses of RSNA to tempol and DETC were significantly greater in spontaneously hypertensive rats than in normotensive rats (n=6, respectively). Local application of sodium nitroprusside (1 mmol/L) or NG-nitro-L-arginine methyl ester (0.11 mol/L) altered neither basal RSNA nor tempol-induced reductions in RSNA (n=6 and 5, respectively). A voltage-gated potassium channel blocker, 4-aminopyridine (0.1 mol/L), significantly decreased basal RSNA (by −81±1%) and completely prevented DETC-induced increases in RSNA (n=5). These results suggest that reactive oxygen species play a role in the regulation of peripheral sympathetic nerve activity, and that at least part of this mechanism is mediated through voltage-gated potassium channels.