Peroxynitrite and the regulation of Na(+),K(+)-ATPase activity by angiotensin II in the rat proximal tubule.

NO reacts spontaneously with superoxide to produce the potent oxidant peroxynitrite. Studies were designed to examine the role of NO-derived oxidants and peroxynitrite on the regulation of Na(+),K(+)-ATPase activity by angiotensin II (ANG II) freshly isolated rat proximal tubules. At picomolar concentrations ANG II stimulates Na(+),K(+)-ATPase activity, but at nanomolar concentrations stimulation is lost. Superoxide dismutase (SOD) was used to examine the role of superoxide and deferoxamine (DFO) and uric acid (UA) were used to examine the role of peroxynitrite. SOD (200 U/mL, 5-min preincubation) restored the stimulatory effect of ANG II (1.31 +/- 0.08-fold; n = 4; P < 0.05 compared to 10(-7) M alone), suggesting a role for superoxide. DFO (100 microm, 5-min preincubation) also restored the stimulatory effect of ANG II (1.40 +/- 0.08-fold; n = 4; P < 0.05, compared to 10(-7) M alone), as did UA (1.22 +/- 0.07-fold; n = 5; P < 0.05, compared to 10(-7) M alone). The NO synthesis inhibitor, N-monomethyl-L-arginine (L-NMMA, 2 mM; 5-min preincubation), also unmasked a stimulatory effect of ANG II at 10(-7) M (1.4 +/- 0.1-fold; n = 7; P < 0.05, compared to 10(-7) M alone). The generation of peroxynitrite was further evidenced by the formation of 3-nitrotyrosine (3-NT). 3-NT increased 3.5-fold in tubules exposed to ANG II (10(-7) M) (0.0054 +/- 0.0019 3-NT/100 tyrosines for control and 0.019 +/- 0.0058 3-NT/100 tyrosines for ANG II, P < .05; n = 4) and L-NMMA prevented the increase. These data suggest that peroxynitrite signaling participates in the regulation of renal of Na(+),K(+)-ATPase activity.     

Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia

Oxidative stress may increase production of superoxide and nitric oxide, leading to formation of prooxidant peroxynitrite to cause vascular dysfunction. Having found nitrotyrosine residues, a marker of peroxynitrite action, in placental vessels of preeclamptic and diabetic pregnancies, we determined whether vasoreactivity is altered in these placentas and treatment with peroxynitrite produces vascular dysfunction. The responses of diabetic, preeclamptic, and normal placentas to increasing concentrations of the vasoconstrictors U-46619 (10(-9)-10(-7) M) and ANG II (10(-9)-10(-7) M) and the vasodilators glyceryl trinitrate (10(-9)-10(-7) M) and prostacyclin (PGI(2); 10(-8)-10(-6) M) were compared as were responses to these agents in normal placentas before and after treatment with 3.16 x 10(-4) M peroxynitrite for 30 min. Responses to both vasoconstrictors and vasodilators were significantly attenuated in diabetic and preeclamptic placentas compared with controls. Similarly, responses to U-46619, nitroglycerin, and PGI(2), but not ANG II, were significantly attenuated following peroxynitrite treatment. The presence of nitrotyrosine residues confirmed peroxynitrite interaction with placental vessels. Overall, our data suggest that peroxynitrite formation is capable of attenuating vascular responses in the human placenta.     

Angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis

The endothelium plays a central role in the maintenance of vascular homeostasis. One of the main effectors of endothelial dysfunction is ANG II, and pharmacological approaches to limit ANG II bioactivity remain the cornerstone of cardiovascular therapeutics. Angiotensin converting enzyme-2 (ACE2) has been identified as a critical negative modulator of ANG II bioactivity, counterbalancing the effects of ACE in determining net tissue ANG II levels; however, the role of ACE2 in the vasculature remains unknown. In the present study, we hypothesized that ACE2 is a novel target to limit endothelial dysfunction and atherosclerosis. To this aim, we performed in vitro gain and loss of function experiments in endothelial cells and evaluated in vivo angiogenesis and atherosclerosis in apolipoprotein E-knockout mice treated with AdACE2. ACE2-deficient mice exhibited impaired endothelium-dependent relaxation. Overexpression of ACE2 in human endothelial cells stimulated endothelial cell migration and tube formation, and limited monocyte and cellular adhesion molecule expression; effects that were reversed in ACE2 gene silenced and endothelial cells isolated from ACE2-deficient animals. ACE2 attenuated ANG II-induced reactive oxygen species production in part through decreasing the expression of p22phox. The effects of ACE2 on endothelial activation were attenuated by pharmacological blockade of ANG-(1-7) with A779. ACE2 promoted capillary formation and neovessel maturation in vivo and reduced atherosclerosis in apolipoprotein E-knockout mice These data indicate that ACE2, in an ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis via a mechanism that may involve attenuation of NADPHox-induced reactive oxygen species production. ACE2-based treatment approaches may be a novel approach to limit aberrant vascular responses and atherothrombosis.     

Effects of chronic dietary salt loading on the renin angiotensin and adrenergic systems of rainbow trout (Oncorhynchus mykiss)

Previous studies have demonstrated that chronic dietary salt loading causes hypertension and a decreased sensitivity of the systemic vasculature to α-adrenergic stimulation and other hypertensive stimuli (e.g. hypercapnia) in rainbow trout (Oncorhynchus mykiss). This reduced sensitivity to hypertensive stimuli is consistent with a possible blunting of homeostatic responses normally aimed at raising blood pressure. To test this idea, we examined the consequences of long-term salt feeding and the associated hypertension on the interactive capacities of the renin angiotensin system (RAS) and adrenergic systems to elevate blood pressure in trout. Secretion of catecholamines in response to a range of doses of homologous ANG II in vivo and in situ (using a perfused posterior cardinal vein preparation) was reduced in the salt-fed fish. The reduced sensitivity to ANG II could not be explained by alterations in stored catecholamine (adrenaline or noradrenaline) levels or the general responsiveness of the chromaffin cells to depolarizing stimuli (60 mmol/l KCl). Despite the decreased responsiveness of the chromaffin cells to ANG II, plasma catecholamines were increased to a greater extent in the salt-fed fish during acute hypoxia (a condition that activates the RAS). Interestingly, the pressor effects of ANG II in vivo were actually heightened in the salt-fed fish. The increased pressor response to exogenous ANG II was likely attributable to its direct interaction with vascular ANG II receptors because the effect persisted even after blockade of α-adrenergic receptors. Treating fish with the vascular smooth muscle relaxant papaverine caused similar reductions in blood pressure and increases in plasma ANG II levels regardless of diet. Similarly, inhibition of angiotensin converting enzyme with lisinopril reduced blood pressure equally in control and salt-fed fish. These results indicate that, while long-term dietary salt loading blunts the response of trout chromaffin cells to ANG II, the RAS itself appears to be unaffected. Indeed, the capacity of ANG II to elevate blood pressure is not compromised nor do fish exhibit a reduced capacity to mount an acute humoral adrenergic stress response during acute hypoxia.     

Interleukin-10 counteracts impaired endothelium-dependent relaxation induced by ANG II in murine aortic rings

ANG II stimulates the production of reactive oxygen species and activates proinflammatory cytokines leading to endothelial dysfunction. We hypothesized that the anti-inflammatory cytokine IL-10 counteracts the impairment in endothelium-dependent ACh relaxation caused by ANG II. Aortic rings of C57BL/6 mice were incubated in DMEM in the presence of vehicle (deionized H(2)O), ANG II (100 nmol/l), recombinant mouse IL-10 (300 ng/ml), or both ANG II and IL-10 for 22 h at 37 degrees C. After incubation, rings were mounted in a wire myograph to assess endothelium-dependent vasorelaxation to cumulative concentrations of ACh. Overnight exposure of aortic rings to ANG II resulted in blunted ACh-induced vasorelaxation compared with that shown in untreated rings (maximal response = 44 +/- 3% vs. 64 +/- 3%, respectively; P<0.05). IL-10 treatment significantly restored this impairment in relaxation (63 +/- 2%). In addition, the NADPH oxidase inhibitor apocynin restored the impairment in relaxation (maximal response = 76 +/- 3%). Western blotting showed increased gp91(phox) expression (a subunit of NADPH oxidase) in response to ANG II. Vessels treated with a combination of ANG II and IL-10 showed decreased expression of gp91(phox). Immunohistochemical analysis showed increased gp91(phox) expression in ANG II-treated vessels compared with those treated with combined ANG II and IL-10. We found that the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation in response to long-term incubation with ANG II via decreasing NADPH oxidase expression.     

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation

Although ANG II exerts a variety of effects on the cardiovascular system, its effects on the peripheral parasympathetic neurotransmission have only been evaluated by changes in heart rate (an effect on the sinus node). To elucidate the effect of ANG II on the parasympathetic neurotransmission in the left ventricle, we measured myocardial interstitial ACh release in response to vagal stimulation (1 ms, 10 V, 20 Hz) using cardiac microdialysis in anesthetized cats. In a control group (n = 6), vagal stimulation increased the ACh level from 0.85 +/- 0.03 to 10.7 +/- 1.0 (SE) nM. Intravenous administration of ANG II at 10 microg x kg(-1) x h(-1) suppressed the stimulation-induced ACh release to 7.5 +/- 0.6 nM (P < 0.01). In a group with pretreatment of intravenous ANG II receptor subtype 1 (AT(1) receptor) blocker losartan (10 mg/kg, n = 6), ANG II was unable to inhibit the stimulation-induced ACh release (8.6 +/- 1.5 vs. 8.4 +/- 1.7 nM). In contrast, in a group with local administration of losartan (10 mM, n = 6) through the dialysis probe, ANG II inhibited the stimulation-induced ACh release (8.0 +/- 0.8 vs. 5.8 +/- 1.0 nM, P < 0.05). In conclusion, intravenous ANG II significantly inhibited the parasympathetic neurotransmission through AT(1) receptors. The failure of local losartan administration to nullify the inhibitory effect of ANG II on the stimulation-induced ACh release indicates that the site of this inhibitory action is likely at parasympathetic ganglia rather than at postganglionic vagal nerve terminals.     

Early gestation dexamethasone programs enhanced postnatal ovine coronary artery vascular reactivity

Excessive exposure of the fetus to maternally derived corticosteroids has been linked to the development of adult-onset diseases. To determine if early gestation corticosteroid exposure alters subsequent coronary artery reactivity, we administered dexamethasone (0.28 mg.kg(-1).day(-1)) to pregnant ewes at 27-28 days gestation (term being 145 days). Vascular responsiveness was assessed in endothelium-intact coronary and mesenteric arteries isolated from steroid-exposed and age-matched control fetal sheep at 123-126 days gestation and lambs at 4 mo of age. Lambs exposed to maternal dexamethasone had higher mean arterial blood pressures than the age-matched controls (93 +/- 3 vs. 83 +/- 5 mmHg, P < 0.05). Mesenteric arteries from the steroid-exposed fetuses displayed diminished responses to ANG II, relative to controls. In 4-mo-old lambs, prenatal dexamethasone exposure significantly increased coronary artery vasoconstriction to ANG II, ACh, and U-46619, but not KCl. In contrast, postnatal mesenteric artery reactivity was unaltered by steroid exposure. Compared with fetal mesenteric reactivity, postnatal mesenteric reactivity to ANG II, phenylephrine, and U-46619 was diminished, whereas the response to 120 mmol/l KCl was heightened. Coronary artery ANG II receptor protein expression was not significantly altered by steroid exposure in either age group. These findings demonstrate that early-gestation glucocorticoid exposure programs postnatal elevations in blood pressure and selectively enhances coronary artery responsiveness to second messenger-dependent vasoconstrictors. Glucocorticoid-induced alterations in coronary vascular smooth muscle structure or function may provide a mechanistic link between an adverse intrauterine environment and later cardiovascular disease.     

Elevated salt intake impairs dilation of rat skeletal muscle resistance arteries via ANG II suppression

Vasodilator responses were assessed in resistance arteries (100-200 microm) isolated from the gracilis muscle of normotensive rats after changes in dietary salt intake. Sprague-Dawley rats were maintained on either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 4-8 wk (chronic) or 3 days (short-term) with water ad libitum. One group of short-term HS rats received a continuous intravenous infusion of a low dose (5 ng x kg(-1) x min(-1)) of ANG II to prevent the ANG II suppression that occurs with HS diet. Short-term and chronic HS diet eliminated arterial dilation in response to ACh and reduced PO(2) (30-40 mmHg) and the stable prostacyclin analog iloprost. ANG II infusion preserved the response to these vasodilator stimuli in short-term HS animals. Dilator responses to sodium nitroprusside and forskolin were unaffected by HS diet. These findings suggest that ANG II suppression during HS diet impairs vascular relaxation mechanisms upstream from the cAMP and cGMP second messenger systems.     

Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system

The renin-angiotensin system is a central component of the physiological and pathological responses of cardiovascular system. Its primary effector hormone, angiotensin II (ANG II), not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The myriad effects of ANG II depend on time (acute vs. chronic) and on the cells/tissues upon which it acts. In addition to inducing G protein- and non-G protein-related signaling pathways, ANG II, via AT₁ receptors, carries out its functions via MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases [PDGF, EGFR, insulin receptor], and nonreceptor tyrosine kinases [Src, JAK/STAT, focal adhesion kinase (FAK)]. AT₁R-mediated NAD(P)H oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis. ANG II also promotes the association of scaffolding proteins, such as paxillin, talin, and p130Cas, leading to focal adhesion and extracellular matrix formation. These signaling cascades lead to contraction, smooth muscle cell growth, hypertrophy, and cell migration, events that contribute to normal vascular function, and to disease progression. This review focuses on the structure and function of AT₁ receptors and the major signaling mechanisms by which angiotensin influences cardiovascular physiology and pathology. vascular smooth muscle; NAD(P)H oxidase; tyrosine and nontyrosine receptor kinases; endothelial dysfunction; vascular disease     

Homocysteine stimulates NADPH oxidase-mediated superoxide production leading to endothelial dysfunction in rats

Elevation of blood homocysteine (Hcy) levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. We previously reported that oxidative stress contributed to Hcy-induced inflammatory response in vascular cells. In this study, we investigated whether NADPH oxidase was involved in Hcy-induced superoxide anion accumulation in the aorta, which leads to endothelial dysfunction during hyperhomocysteinemia. Hyperhomocysteinemia was induced in rats fed a high-methionine diet. NADPH oxidase activity and the levels of superoxide and peroxynitrite were markedly increased in aortas isolated from hyperhomocysteinemic rats. Expression of the NADPH oxidase subunit p22 phox increased significantly in these aortas. Administration of an NADPH oxidase inhibitor (apocynin) not only attenuated aortic superoxide and peroxynitrite to control levels but also restored endothelium-dependent relaxation in the aortas of hyperhomocysteinemic rats. Transfection of human endothelial cells or vascular smooth muscle cells with p22 phox siRNA to inhibit NADPH oxidase activation effectively abolished Hcy-induced superoxide anion production, thus indicating the direct involvement of NADPH oxidase in elevated superoxide generation in vascular cells. Taken together, these results suggest that Hcy-stimulated superoxide anion production in the vascular wall is mediated through the activation of NADPH oxidase, which leads to endothelial dysfunction during hyperhomocysteinemia.     

Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II

Our purpose was to address the role of NAPDH oxidase-derived superoxide anion in the vascular response to ANG II. Blood pressure, aortic superoxide anion, 3-nitrotyrosine, and medial cross-sectional area were compared in wild-type mice and in mice that overexpress human superoxide dismutase (hSOD). The pressor response to ANG II was significantly less in hSOD mice. Superoxide anion levels were increased twofold in ANG II-treated wild-type mice but not in hSOD mice. 3-Nitrotyrosine increased in aortic endothelium and adventitia in wild-type but not hSOD mice. In contrast, aortic medial cross-sectional area increased 50% with ANG II in hSOD mice, comparable to wild-type mice. The lower pressor response to ANG II in the mice expressing hSOD is consistent with a pressor role of superoxide anion in wild-type mice, most likely because it reacts with nitric oxide. Despite preventing the increase in superoxide anion and 3-nitrotyrosine, the aortic hypertrophic response to ANG II in vivo was unaffected by hSOD.     

The role of inducible nitric oxide synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats

Hemorrhagic shock (HS) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, NO has been implicated to cause hyporesponsiveness of blood vessel in vitro towards vasoconstrictors in refractory (decompensated) HS. In our in vivo model, we examined the effects of aminoguanidine (AG), a known iNOS inhibitor, with angiotensin II (ANG II), a vasoconstrictor, following hemorrhagic shock decompensatory phase (HSDP) on percentage survival, vascular responsiveness, mean arterial blood pressure (MABP), heart rate and mean nitrate/nitrite levels in anaesthetized rats. HSDP (3 h) was achieved via constant pressure method (40-45 mmHg). MABP and heart rate was measured via the left carotid artery. Plasma collected from HSDP rats was used to measure nitrate/nitrite levels. Vascular hyporeactivity to ANG II was carried out using HSDP aortic strips, precontracted with KCl and noradrenaline. Sham-operated rats served as controls. HSDP rats decreased percentage survival, vascular contractility to ANG II and noradrenaline, MABP, heart rate while showing increased levels of nitrate/nitrite. Infusion of AG with ANG II, increased percentage survival and had reversed these cardiovascular effects of HSDP rats. This study indicates that excessive NO formation from iNOS activity induces vascular hyporeactivity and decompensation in HSDP. This might suggest that selective NOS inhibitor, AG, when coupled with ANG II, show reduction in NO's effect in HSDP.     

Central effects of angiotensin II in conscious hamsters: drinking,pressor response,and release of vasopressin

Summary The effects of intracerebroventricular (icv) injections of angiotensin II (ANG II) on water intake, blood pressure, heart rate, and plasma arginine-vasopressin (AVP) concentration were studied in chronically instrumented adult male Syrian golden hamsters (Mesocricetus auratus). Furthermore, the effects of pharmacological ganglionic blockade, and of vascular AVP receptor blockade, on central ANG II-induced cardiovascular responses were investigated. ANG II (1, 10, and 100 ng, icv) elicited dose-dependent increases in water intake and arterial blood pressure. Heart rate showed a biphasic response with a short initial non dose-dependent tachycardic and a subsequent longer lasting bradycardic phase. Plasma AVP concentration was increased two and a half fold with 100 ng ANG II icv. Both ganglionic blockade and vascular AVP receptor blockade significantly attenuated the central ANG II-induced pressor response. The tachycardic phase of the heart rate response was abolished by ganglionic blockade and the bradycardic phase was significantly diminished by AVP receptor blockade. The results support the hypothesis that brain ANG II may participate in the central control of body fluid volume and in central cardiovascular regulation in conscious hamsters.     

Role of endothelium-derived relaxing factors in the renal response to vasoactive agents in hypothyroid rats

This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the abnormal renal vascular reactivity of hypothyroid rats. Renal responses to vasoconstrictors [VC: phenylephrine (PHE) and ANG II] and vasodilators [VD: ACh, sodium nitroprusside (SNP), and papaverine (PV)] were studied in kidneys from control and hypothyroid rats under normal conditions and after NO or EDHF blockade. NO was blocked by the administration of Nomega-nitro-l-arginine methyl ester (l-NAME) and EDHF by the administration of tetraethylammonium (TEA) or by an increased extracellular K+. The response to VC was also evaluated after endothelium removal. Hypothyroid kidneys showed reduced responsiveness to PHE and a normal response to ANG II. l-NAME and TEA administration produced an increased sensitivity to PHE and to ANG II in control preparations. l-NAME also increased the response to PHE in hypothyroid kidneys, but the differences between control and hypothyroid kidneys were maintained. TEA administration did not change the response to either VC in hypothyroid preparations. In endothelium-removed preparations, TEA was unable to increase pressor responsiveness to VC. Hypothyroid kidneys showed reduced responsiveness to ACh and SNP and normal response to PV. The differences between hypothyroid and control preparations in the responses to ACh and SNP were maintained after l-NAME or increased K+. In conclusion, this study shows that 1) the attenuated response to PHE in hypothyroidism is not related to an increased production of endothelium-derived relaxing factors NO and EDHF; 2) the response to VC in hypothyroid preparations is insensitive to EDHF blockade; and 3) hypothyroid preparations have a reduced reactivity to the NO donor, and NO-independent vasodilatation remains unaffected.     

Coronary arteriolar vasoconstriction to angiotensin II is augmented in prediabetic metabolic syndrome via activation of AT1 receptors

The metabolic syndrome is associated with activation of the renin-angiotensin system. However, whether the coronary vascular response to ANG II is altered under this condition is unknown. Experiments were conducted in control and chronically high-fat-fed dogs with the prediabetic metabolic syndrome both in vitro (isolated coronary arterioles, 60-110 microm) and in vivo (anesthetized and conscious). We found that plasma renin activity and ANG II levels are elevated in high-fat-fed dogs and that this increase in ANG II is associated with a significant increase in ANG II-mediated coronary vasoconstriction in isolated coronary arterioles and in anesthetized open-chest dogs. The vasoconstriction to ANG II is abolished by ANG II type 1 (AT1) receptor blockade. In conscious chronically instrumented dogs, AT1 receptor blockade with telmisartan improved the balance between coronary blood flow and myocardial oxygen consumption in the high-fat-fed dogs but not in normal control dogs, i.e., the relationship between coronary venous Po2 and myocardial oxygen consumption was shifted upward, toward normal control values. Quantitative assessment of coronary arteriolar AT1 and ANG II type 2 (AT2) receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs; however, Western blot analysis showed a significant increase in AT1 receptor protein level with no change in AT2 receptor protein density. These findings indicate that AT1 receptor-mediated coronary constriction is augmented in the prediabetic metabolic syndrome and contributes to impaired control of coronary blood flow via increases in circulating ANG II and/or coronary arteriolar AT1 receptor density.     

AT1 receptor-mediated augmentation of angiotensinogen, oxidative stress, and inflammation in ANG II-salt hypertension

Augmentation of intrarenal angiotensinogen (AGT) synthesis, secretion, and excretion is associated with the development of hypertension, renal oxidative stress, and tissue injury during ANG II-dependent hypertension. High salt (HS) exacerbates hypertension and kidney injury, but the mechanisms remain unclear. In this study, we determined the consequences of HS intake alone compared with chronic ANG II infusion and combined HS plus ANG II on the stimulation of urinary AGT (uAGT), renal oxidative stress, and renal injury markers. Sprague-Dawley rats were subjected to 1) a normal-salt diet [NS, n = 5]; 2) HS diet [8% NaCl, n = 5]; 3) ANG II infusion in NS rats [ANG II 80 ng/min, n = 5]; 4) ANG II infusion in HS rats [ANG II+HS, n = 5]; and 5) ANG II infusion in HS rats treated with ANG II type 1 receptor blocker (ARB) [ANG II+HS+ARB, n = 5] for 14 days. Rats fed a HS diet alone did not show changes in systolic blood pressure (SBP), proteinuria, cell proliferation, or uAGT excretion although they did exhibit mesangial expansion, collagen deposition, and had increased NADPH oxidase activity accompanied by increased peroxynitrite formation in the kidneys. Compared with ANG II rats, the combination of ANG II infusion and a HS diet led to exacerbation in SBP (175 ± 10 vs. 221 ± 8 mmHg; P < 0.05), proteinuria (46 ± 7 vs. 127 ± 7 mg/day; P < 0.05), and uAGT (1,109 ± 70 vs.. 7,200 ± 614 ng/day; P < 0.05) associated with greater collagen deposition, mesangial expansion, interstitial cell proliferation, and macrophage infiltration. In both ANG II groups, the O(2)(-) levels were increased due to increased NADPH oxidase activity without concomitant increases in peroxynitrite formation. The responses in ANG II rats were prevented or ameliorated by ARB treatment. The results indicate that HS independently stimulates ROS formation, which may synergize with the effect of ANG II to limit peroxynitrite formation, leading to exacerbation of uAGT and greater injury during ANG II salt hypertension.     

Angiotensin II and tumor necrosis factor-alpha synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-kappaB, p38, and reactive oxygen species

We examined whether ANG II and TNF-alpha cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-alpha stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-kappaB-dependent pathway. In contrast, TNF-alpha-induced MCP-1 expression was potently suppressed by blockade of NF-kappaB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-alpha-induced activation of NF-kappaB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-alpha-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-alpha cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-alpha further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-alpha alone. These results suggested that ANG II and TNF-alpha synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFkappaB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-alpha cooperatively stimulate vascular inflammation in vivo as well as in vitro.     

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants.The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM).In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.