Effects of angiotensin II AT1-receptor blockade on high fat diet-induced vascular oxidative stress and endothelial dysfunction in Dahl salt-sensitive rats.

We examined the effects of angiotensin II AT1-receptor blockade with olmesartan on high fat (HF) diet-induced vascular oxidative stress and endothelial dysfunction in normal salt (NS) diet-fed Dahl salt-sensitive (DSS) rats. Treatment with NS + HF diet (32% crude fat, 0.3% NaCl) for 20 weeks significantly increased blood pressure in DSS rats. NS + HF diet-fed DSS rats also showed higher plasma levels of thiobarbituric acid-reactive substances, aortic superoxide production, and mRNA levels of p22(phox) and gp91(phox) in aortic tissues than NS diet-fed DSS rats. Furthermore, acetylcholine-induced vasorelaxation of aorta from NS + HF diet-fed DSS rats was significantly reduced. In NS + HF diet-fed DSS rats, treatment with olmesartan medoxomil (10 mg/kg per day, p.o.) and hydralazine (25 mg/kg per day, p.o.) similarly decreased blood pressure. However, in these animals, only olmesartan normalized plasma levels of thiobarbituric acid-reactive substances, vascular superoxide in aortic tissues, and acetylcholine-induced vasorelaxation. These data indicate that HF diet-induced hypertension is associated with vascular oxidative stress and endothelial dysfunction in NS diet-treated DSS rats. Inhibition of angiotensin II AT1 receptors may elicit beneficial effects on HF-induced hypertension and vascular injury in subjects that have genetically enhanced sodium-sensitive blood pressure.

Effects of adrenomedullin on cardiac oxidative stress and collagen accumulation in aldosterone-dependent malignant hypertensive rats.

We examined the effects of adrenomedullin on cardiac oxidative stress and collagen accumulation in aldosterone-dependent malignant hypertensive rats. Spontaneously hypertensive rats (SHRs) were treated with one of the following combinations for 4 weeks: tap water and vehicle [0.5% ethanol, subcutaneously (s.c.), n = 5], 1% NaCl in drinking water and vehicle (n = 8), 1% NaCl and aldosterone (0.75 microg/h s.c., n = 8), and 1% NaCl, aldosterone, and adrenomedullin (1.3 microg/kg/h s.c., n = 8). Systolic blood pressure (SBP) and left ventricular (LV) weight were higher in aldosterone-treated SHRs than vehicle- or vehicle/1% NaCl-treated SHRs. Thiobarbituric acid reactive substances (TBARS) levels and NADPH oxidase activity in LV tissues of aldosterone-treated SHRs were also higher than those of vehicle- or vehicle/1% NaCl-treated SHRs, and these changes were associated with increases in LV mRNA levels of p22phox, gp91phox, fibronectin, collagen types I and III, as well as collagen content. Treatment with adrenomedullin did not alter SBP or LV weight but attenuated aldosterone-induced increases in TBARS levels, NADPH oxidase activity, and mRNA levels of p22phox, gp91phox, fibronectin, collagen types I and III, as well as collagen content in LV tissues. These data suggest that NADPH oxidase-mediated reactive oxygen species production is involved in the pathogenesis of cardiac collagen accumulation in aldosterone-dependent malignant hypertensive rats and that the cardioprotective effects of adrenomedullin are 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 F2alpha 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.

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-beta (TGF-beta), 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-beta, 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.

Renal interstitial fluid ATP responses to arterial pressure and tubuloglomerular feedback activation during calcium channel blockade.

A close relationship between changes in renal interstitial fluid (RIF) ATP concentrations and renal autoregulatory or tubuloglomerular feedback (TGF)-dependent changes in renal vascular resistance (RVR) has been demonstrated, but it has not been determined whether the changes in RIF ATP are a consequence or the cause of the changes in RVR. The present study was performed in anesthetized dogs to assess the changes in RIF ATP following changes in renal arterial pressure (RAP) or stimulation of the TGF mechanism under conditions where changes in RVR were prevented by nifedipine, a calcium channel blocker. RIF ATP levels were measured by using microdialysis probes. Intra-arterial infusion of nifedipine (0.36 microg x kg(-1) x min(-1)) increased renal blood flow (RBF: from 4.49 +/- 0.27 to 5.34 +/- 0.39 ml x min(-1) x g(-1)) and glomerular filtration rate (GFR: from 0.84 +/- 0.07 to 1.09 +/- 0.11 ml x min(-1) x g(-1)). Under conditions of nifedipine infusion, autoregulatory adjustments in RBF, GFR, and RVR were not observed during stepwise reductions in RAP within the autoregulatory range (from 135 +/- 7 to 76 +/- 1 mmHg, n = 7). Furthermore, stimulation of the TGF mechanism with intra-arterial infusion of acetazolamide (100 microg x kg(-1) x min(-1)) did not alter RBF, GFR, and RVR (n = 7). During treatment with nifedipine, RIF ATP levels were significantly decreased in response to reductions in RAP (10.7 +/- 0.7, 5.8 +/- 0.7 and 2.8 +/- 0.3 nmol/l at 135 +/- 7, 101 +/- 4, and 76 +/- 1 mmHg, n = 7) and increased by acetazolamide infusion (from 8.8 +/- 0.8 to 17.0 +/- 1.8 nmol/l, n = 7). These results are similar to those that occurred in dogs not treated with nifedipine and thus demonstrate that the changes in RIF ATP can occur in the absence of autoregulatory or TGF-mediated changes in RVR. The data provide further support to the hypothesis that RIF ATP contributes to adjustments in RVR associated with renal autoregulation and changes in activity of the TGF mechanism.

Effects of calcium channel blockade on angiotensin II-induced peritubular ischemia in rats.

Recent studies have indicated that derangement of peritubular capillary (PTC) circulation with consequent tubulointerstitial hypoxia plays a pivotal role in the pathogenesis of renal injury. The present study was performed to determine whether azelnidipine, a new dihydropyridine calcium channel blocker, attenuates angiotensin II (AngII)-induced peritubular ischemia in anesthetized rats. The superficial PTCs were visualized directly using an intravital fluorescence videomicroscope system, and the PTC blood flow was evaluated by analyzing the velocity of fluorescein isothiocyanate-labeled erythrocytes. Intravenous infusion of AngII (50 ng/kg/min, 10 min) significantly increased mean arterial pressure (MAP) and renal vascular resistance (RVR) (by 35 +/- 3% and 110 +/- 32%, respectively), and decreased total renal blood flow (RBF) and PTC erythrocyte velocity (by -34 +/- 4 and -37 +/- 1%, respectively). Treatment with azelnidipine (5 microg/kg/min i.v., 10 min) had no effect on basal MAP, RBF, RVR, or PTC erythrocyte velocity. However, azelnidipine markedly attenuated the AngII-induced increases in MAP (7 +/- 3%) and RVR (40 +/- 4%) and decreases in RBF (-24 +/- 1%) and PTC erythrocyte velocity (-22 +/- 1%). Similar attenuation in the AngII-induced responses of MAP, RBF, RVR, and PTC erythrocyte velocity were observed in rats treated with a higher dose of azelnidipine (20 microg/kg/min i.v., 10 min), which significantly decreased basal MAP and RVR and increased RBF and PTC erythrocyte velocity. These data suggest that calcium channel blockade attenuates AngII-induced peritubular ischemia, which may be involved in its beneficial effects on renal injury.

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.

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 micromol/L), a selective MR antagonist, or PD98059 (10 micromol/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.

Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs: role of adenosine and adenine nucleotides.

AIM: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury. METHODS: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902), respectively. The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology. RESULTS: One 10-min IPC, adenosine A1 receptor antagonist (KW-3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP). Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia. All of these recovery times were dependent on the cycles of 10-min IPC. The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively. CONCLUSION: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively. Renal IPC can protect against ischemia-reperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.

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.

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 [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 (DeltaPsi(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 DeltaPsi(M) and redox-sensitive activation of MAP kinase; however, generated ROS from mitochondria do not contribute to Ang II-induced vasoconstriction.

Effects of a new calcium channel blocker, azelnidipine, on systemic hemodynamics and renal sympathetic nerve activity in spontaneously hypertensive rats.

Antihypertensive treatment with dihydropyridine calcium channel blockers elicits sympathetic nerve activation, which may contribute to cardiovascular events. However, recent clinical studies showed that treatment with azelnidipine, a new dihydropyridine calcium channel blocker, significantly reduced blood pressure in hypertensive patients while either maintaining or actually decreasing heart rate (HR). In this study, we examined the effects of azelnidipine and amlodipine on systemic hemodynamics and renal sympathetic nerve activity (RSNA) in anesthetized spontaneously hypertensive rats (SHR). We also examined the effects of these agents on baroreflex functions by infusing phenylephrine (30 microg/kg/min, i.v.) and sodium nitroprusside (10 microg/kg/min, i.v.) into azelnidipine- or amlodipine-treated SHR. Fifty min after administration of azelnidipine (10 microg/kg/min for 10 min, i.v.), mean arterial pressure (MAP) significantly decreased from 153+/-5 to 122+/-5 mmHg; however, HR and integrated RSNA did not change significantly (from 352+/-9 to 353+/-10 beats/ min and 115+/-5% of baseline, respectively). Infusion of amlodipine (50 microg/kg/min for 10 min) elicited similar effects on MAP (from 152+/-5 to 120+/-4 mmHg). However, amlodipine significantly increased HR (from 351+/-9 to 375+/-11 beats/min) and integrated RSNA (165+/-5% of baseline). Analyses of baroreflex function curves revealed that azelnidipine-treated rats showed a smaller baroreflex function than amlodipine-treated rats (p<0.05). These data suggest that azelnidipine possesses sympathoinhibitory effects, which may be one reason why it had less pronounced effects on HR in hypertensive patients.

Cardiac oxidative stress in acute and chronic isoproterenol-infused rats.

OBJECTIVE: Sympathetic nervous system activity in the myocardium is increased in patients with heart failure. However, the in vivo mechanisms responsible for beta-adrenoceptor-mediated cardiac hypertrophy or remodeling remain unclear. This study aimed to clarify the role of reactive oxygen species (ROS) in mitogen-activated protein (MAP) kinase activation and tissue remodeling of the heart of isoproterenol (ISO)-infused rats. METHODS AND RESULTS: Different doses of ISO (up to 1000 ng/kg/min) were given intravenously to conscious rats for 30 min. Phosphorylated MAP kinase levels (ERK1/2, JNK, p38) and lipid peroxidation were measured in the cardiac left ventricle, revealing the dose-dependent augmentation of MAP kinase phosphorylation and increased lipid peroxidation levels. Simultaneous treatment with 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (Tempol), a membrane-permeable radical scavenger, completely eliminated the increases of phosphorylated MAP kinases and their upstream elements (Raf-1, Rac-1, ASK-1) as well as the increases of cardiac lipid peroxidation induced by the highest dose of ISO infusion. In chronically ISO-infused rats (3 mg/kg/day, s.c. for 10 days), cardiac hypertrophy developed with accompanying increases of collagen content, whereas cardiac phosphorylated MAP kinases returned to normal. Tempol treatment prevented increases of collagen accumulation and type I collagen mRNA without any significant reduction of cardiac mass enlargement induced by chronic ISO infusion. CONCLUSION: 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.

Time-dependent transition of tempol-sensitive reduction of blood pressure in angiotensin II-induced hypertension.

OBJECTIVE: Reactive oxygen species (ROS) participate in the intracellular signalling of angiotensin II. However, the mechanisms of the interaction of ROS with hypertension and mitogen-activated protein kinase (MAPK) in vivo have remained unclear. Angiotensin II infusion provokes sustained hypertension accompanied with enhancement of ROS production; initially hypertension is non-sensitive to ROS, but thereafter becomes sensitive. We examined the time-dependent transition of ROS-sensitive vasoconstriction during angiotensin II infusion and also ROS sensitivity to cardiovascular MAPK activation in acutely and chronically angiotensin II-infused rats. METHODS AND RESULTS: During infusion of a pressor dose of angiotensin II to conscious Sprague-Dawley rats, tempol, a superoxide dismutase mimetic, was administered at 10 min, some 1, 3, 6, 12 and 24 h after the start of infusion. The magnitude of the reduction in blood pressure by tempol was initially negligible, but gradually enlarged, and reached a maximum of 96% of delta increase by angiotensin II at 12 h. However, even after sensitization to tempol, superimposed angiotensin II enabled an increase of blood pressure under tempol treatment. In chronically angiotensin II-infused rats, superimposed angiotensin II exhibited tempol quenchable MAPK activation. CONCLUSIONS: These results indicate that the mechanisms of angiotensin II-induced vasoconstriction may shift from being non-sensitive to ROS to sensitive within 12 h; nevertheless, both ROS non-sensitive vasoconstriction and ROS-sensitive MAPK activation by angiotensin II, which are both seen in the acute phase of infusion, are restored in the late maintaining phase of prolonged angiotensin II infusion.

Blood flow-dependent changes in intrarenal nitric oxide levels during anesthesia with halothane or sevoflurane.

We previously demonstrated that intrarenal nitric oxide (NO) levels and renal blood flow are reduced during halothane anesthesia. Studies were performed to determine if volatile anesthetics-induced reductions in renal NO levels are associated with blood flow changes. Halothane and sevoflurane at 0.8 and 2.4 Mac were administered by inhalation to dogs, and cGMP and NOx concentrations in the renal interstitial fluid were measured by a microdialysis method. Neither halothane nor sevoflurane at 0.8 Mac altered renal blood flow and renal interstitial cyclic guanosine monophosphate (cGMP) and NOx levels, but both anesthetics significantly decreased these values at 2.4 Mac. Using an adjustable aortic clamp, renal perfusion pressure was reduced in 2 steps without halothane and sevoflurane anesthesia. Renal blood flow as well as cGMP and NOx concentrations in the renal interstitial fluid were unchanged within the autoregulatory range, but significantly decreased below the autoregulatory range. Changes in cGMP and NOx concentrations in the renal interstitial fluid were highly correlated with renal blood flow changes during halothane or sevoflurane anesthesia, and during stepwise reductions in renal perfusion pressure. The results suggested that halothane- and sevoflurane-induced decreases in intrarenal NO levels result from reductions in blood flow.

Olmesartan improves endothelin-induced hypertension and oxidative stress in rats.

Recent studies have indicated that both endothelin (ET) and angiotensin (Ang) II stimulate oxidative stress, which contributes to the development of hypertension. Here, we examined the effects of Ang II type 1 (AT1) receptor blockade on reactive oxygen species (ROS) formation in ET-dependent hypertension. Chronic ET-1 infusion (2.5 pmol/kg/min, i.v., n=7) into rats for 14 days increased systolic blood pressure from 113+/-1 to 141+/-2 mmHg. ET-1-infused rats showed greater plasma renin activity (8.1+/-0.8 Ang I/ml/h), and greater Ang I (122+/-28 fmol/ml) and Ang II levels (94+/-13 fmol/ml) than vehicle (0.9% NaCl)-infused rats (3.1+/-0.6 Ang I/ml/h, 45+/-8 and 47+/-7 fmol/ml, respectively, n=6). Angiotensin converting enzyme and AT1 receptor expression in aortic tissues were similar between the vehicle- and ET-1-infused rats. Vascular superoxide anion (O2-) production and plasma thiobarbituric acid-reactive substance (TBARS) levels were greater in ET-1-infused rats (27+/-1 counts per minutes [CPM]/mg dry tissue weight and 8.9+/-0.8 micromol/l, respectively) than vehicle-infused rats (16+/-1 CPM/mg and 5.1+/-0.1 micromol/l, respectively). The ET-1-induced hypertension was prevented by simultaneous treatment with a new AT1 receptor antagonist, olmesartan (0.01% in chow, 117+/-5 mmHg, n =7), or hydralazine (15 mg/kg/day in drinking water, 118+/-4 mmHg, n=6). Olmesartan prevented ET-1-induced increases in vascular O2- production (15+/-1 CPM/mg) and plasma TBARS (5.0+/-0.1 micromol/l). Vascular O2- production and plasma TBARS were also decreased by hydralazine (21+/-1 CPM/mg and 7.0+/-0.3 micromol/l, respectively), but these levels were significantly higher than in vehicle-infused rats. These data suggest that ET-dependent hypertension is associated with augmentation of Ang II levels and ROS formation. The combined effects of the elevations in circulating ET-1 and Ang II, as well as the associated ROS production, may contribute to the development of hypertension induced by chronic ET-1 infusion.

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 microL, 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 microL, 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 N(G)-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.

Angiotensin II stimulates superoxide production via both angiotensin AT1A and AT1B receptors in mouse aorta and heart.

The present study was conducted to determine the roles of angiotensin AT(1A) and AT(1B) receptors in angiotensin II-induced superoxide anion production in mouse aorta and heart. Superoxide anion production in aorta was determined by the lucigenin chemiluminescence method, and thiobarbituric acid reactive substances in heart tissues were measured by biochemical assay. The basal production rate of superoxide anion in aorta of wild type (WT) mice was significantly higher than in angiotensin AT(1A) receptor knockout (AT(1A) KO) mice. Angiotensin II (2.8 mg/kg/day, s.c. for 13 days) significantly increased superoxide anion production in aorta of both AT(1A) KO and WT mice. However, the superoxide anion production rate in aorta of angiotensin II-infused AT(1A) KO mice was significantly lower than in angiotensin II-infused WT mice. Valsartan (40 mg/kg/day in drinking water) prevented angiotensin II-induced superoxide anion production in aorta of WT and AT(1A) KO mice. Similarly, thiobarbituric acid reactive substances levels in heart tissues of angiotensin II-treated WT and AT(1A) KO mice were significantly higher than those in vehicle-infused WT and AT(1A) KO mice, respectively. Valsartan prevented angiotensin II-induced increases of thiobarbituric acid reactive substances levels in heart tissue of both WT and AT(1A) KO mice. These results indicate that angiotensin II stimulates superoxide anion production via both angiotensin AT(1A) and AT(1B) receptors, and that angiotensin AT(1A) receptors appear to play a predominant role in angiotensin II-induced superoxide anion production in mouse aorta and heart.

Effects of AT1 receptor blockade on renal injury and mitogen-activated protein activity in Dahl salt-sensitive rats.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an important intracellular mediator of angiotensin II (Ang II)-induced cell growth and differentiation. Here, we examined the effect of angiotensin II type 1 receptor (AT1) receptor blockade on renal injury and MAPK activity in Dahl salt-sensitive (DS) rats. METHODS: DS rats were maintained on a high (H: 8.0%NaCl, N= 8) or low (L: 0.3%NaCl, N= 7) salt diet, or H + candesartan cilexetil (10 to 15 mg/kg/day, N= 8). Urinary protein excretion (UproteinV), renal cortical collagen content, and glomerular injury (assessed by semiquantitative morphometric analysis) were determined after 4-week treatments. Plasma and kidney Ang II levels were measured by radioimmunoassay. Protein levels of AT1 and AT2 receptors in the renal cortical tissues were analyzed by Western-blotting analyses. MAPKs activities, including extracellular signal-regulated kinases (ERK)1/2, c-Jun NH2-terminal kinases (JNK), p38 MAPK, and Big-MAPK-1 (BMK1), were measured by Western-blotting analyses or in vitro kinase assays. RESULTS: DS/H rats showed higher mean blood pressure (MBP), UproteinV, and renal cortical collagen content than DS/L rats. Increased ERK1/2, JNK, and BMK1 activities were observed in renal cortical tissues of DS/H rats (approximately 6.3-, 4.5-, and 2.5-fold, respectively), whereas p38 MAPK activity was unchanged. Plasma Ang II levels were significantly reduced in DS/H rats compared with DS/L rats, whereas kidney Ang II contents and AT1 receptor protein levels were similar. Candesartan did not alter MBP, but significantly reduced UproteinV and collagen content, and ameliorated progressive sclerotic and proliferative glomerular changes. Furthermore, candesartan decreased renal tissue Ang II contents (from 216 +/- 19 to 46 +/- 3 fmol/mL) and ERK1/2, JNK, and BMK1 activities (-45%, -60%, and -70%, respectively) in DS/H rats. CONCLUSION: In DS hypertensive rats, some of the renoprotective effects of AT1 receptor blockade are accompanied by reductions in intrarenal Ang II contents and MAPK activity, which might not be mediated through arterial pressure changes.

Role of interstitial ATP and adenosine in the regulation of renal hemodynamics and microvascular function.

The role of adenosine in the regulation of renal hemodynamics and function has been studied extensively; however, another purine agent, ATP, is also gaining recognition for its paracrine role in the kidney. Adenosine and ATP bind to specific membrane-bound P1 and P2 purinoceptors, respectively, and initiate a variety of biological effects on renal microvascular tone, mesangial cell function, and renal epithelial transport. The purpose of this review is to summarize the potential roles of interstitial ATP and adenosine as regulators of renal hemodynamics and microcirculation. In vitro blood-perfused juxtamedullary nephron preparation was used to assess the roles of ATP and adenosine in the regulation of renal microvascular tone. This approach mimics the adventitial exposure of renal microvascular smooth muscle to ATP and adenosine synthesized locally and released into the interstitial fluid. ATP selectively vasoconstricts afferent but not efferent arterioles via P2X and P2Y receptors, whereas, adenosine vasoconstricts both vascular segments via activation of adenosine A(1) receptors. Furthermore, selective P2X and P2Y receptor stimulation increases intracellular calcium concentration in vascular smooth muscle cells that are freshly isolated from the preglomerular microvasculature. These data support the hypothesis that interstitial ATP plays a critical role in the control of renal microvascular function through mechanisms that are independent of adenosine receptors. We have recently developed a renal microdialysis method to determine the dynamics of ATP and adenosine levels in the renal cortical interstitium. In this review, we also summarize current knowledge pertaining to the alterations in renal interstitial ATP and adenosine in some pathophysiological conditions.