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Normal pregnancy is associated with adaptive hemodynamic, hormonal, and vascular changes, and estrogen (E2) may promote vasodilation during pregnancy; however, the specific E2 receptor (ER) subtype, post-ER signaling mechanism, and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptations involve changes in the expression/distribution/activity of distinct ER subtypes in a blood vessel-specific manner. Blood pressure (BP) and plasma E2 were measured in virgin and pregnant (day 19) rats, and the thoracic aorta, carotid artery, mesenteric artery, and renal artery were isolated for measurements of ERα, ERβ, and G protein-coupled receptor 30 [G protein-coupled ER (GPER)] expression and tissue distribution in parallel with relaxation responses to E2 (all ERs) and the specific ER agonist 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERα), diarylpropionitrile (DPN; ERβ), and G1 (GPER). BP was slightly lower and plasma E2 was higher in pregnant versus virgin rats. Western blots revealed increased ERα and ERβ in the aorta and mesenteric artery and GPER in the aorta of pregnant versus virgin rats. Immunohistochemistry revealed that the increases in ERs were mainly in the intima and media. In phenylephrineprecontracted vessels, E2 and PPT caused relaxation that was greater in the aorta and mesenteric artery but similar in the carotid and renal artery of pregnant versus virgin rats. DPN- and G1-induced relaxation was greater in the mesenteric and renal artery than in the aorta and carotid artery, and aortic relaxation to G1 was greater in pregnant versus virgin rats. The nitric oxide synthase inhibitor Nϖ-nitro-L-arginine methyl ester with or without the cyclooxygenase inhibitor indomethacin with or without the EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT, and G1-induced relaxation in the aorta of pregnant rats, suggesting an endothelium-dependent mechanism, but did not affect E2-, PPT-, DPN-, or G1-induced relaxation in other vessels, suggesting endothelium-independent mechanisms. E2, PPT, DPN, and G1 caused relaxation of Ca2+ entry-dependent KCl contraction, and the effect of PPT was greater in the mesenteric artery of pregnant versus virgin rats. Thus, during pregnancy, an increase in ERα expression in endothelial and vascular smooth muscle layers of the aorta and mesenteric artery is associated with increased ERα-mediated relaxation via endothelium-derived vasodilators and inhibition of Ca2+ entry into vascular smooth muscle, supporting a role of aortic and mesenteric arterial ERα in pregnancy-associated vasodilation. GPER may contribute to aortic relaxation while enhanced ERβ expression could mediate other genomic vascular effects during pregnancy. [ABSTRACT FROM AUTHOR]

Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor fork-head box 0 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahi salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, H2O2 dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO 1 expression with small-interfering RNA. H2O2 rapidly induced FOXO 1 nuclear translocation, but the effect was blocked by c-Jun NH2-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed H2O2's effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to test whether morphine prevents the mitochondrial permeability transition pore (mPTP) opening through Zn2+ and glycogen synthase kinase 3β (GSK-3β). Fluorescence dyes including Newport Green Dichiorofluorescein (DCF), 4-amino-5-methylamino2',7'-difluorofluorescein (DAF-FM), and tetramethyirhodamine ethyl ester (TMRE) were used to image free Zn2+ nitric oxide (NO), and mitochondrial membrane potential (ΔΨm), respectively. Fluorescence images were obtained with confocal microscopy. Cardiomyocytes treated with morphine for 10 mm showed a significant increase in Newport Green DCF fluorescence intensity, an effect that was reversed by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), indicating that morphine mobilizes Zn2+ via NO. Morphine rapidly produced NO. ODQ and NS2028, the inhibitors of guanylyl cyclase, prevented Zn2+ release by morphine, implying that cGMP is involved in the action of morphine. The effect of morphine on Zn2+ release was also abolished by KT5823, a specific inhibitor of protein kinase G (PKG). Morphine prevented oxidant-induced loss of ΔΨm, indicating that morphine can modulate the mPTP opening. The effect of morphine on the mPTP was reversed by KT5823 and the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). The action of morphine on the mPTP was lost in cells transfected with the constitutively active GSK-3β mutant, suggesting that morphine may prevent the mPTP opening by inactivating GSK3β. In support, morphine significantly enhanced phosphorylation of GSK-3β at Ser9, and this was blocked by TPEN. GSK-3β small interfering RNA prevented the pore opening in the control cardiomyocytes but failed to enhance the effect of morphine on the mPTP opening. In conclusion, morphine mobilizes intracellular Zn2+ through the NO/cGMP/PKG signaling pathway and prevents the mPTP opening by inactivating GSK-3β through Zn2+. [ABSTRACT FROM AUTHOR]

Inorganic nitrite has recently been recognized to possess vascular activity that is enhanced in hypoxia. This has been demonstrated in humans in the forearm vascular bed. In animal models nitrite reduces pulmonary vascular resistance, but its effects upon the pulmonary circulation of humans have not yet been demonstrated. This paradigm is of particular interest mechanistically since the pulmonary vasculature is known to behave differently to the systemic. To investigate, 18 healthy volunteers were studied in a hypoxic chamber (inspired oxygen, 12%) or while breathing room air. Each received an infusion of sodium nitrite (1 μmol/min) or 0.9% saline. Three protocols were performed: nitrite! hypoxia (n = 12), saline/hypoxia (n = 6), and nitrite/normoxia (n = 6). Venous blood was sampled for plasma nitrite, forearm blood flow was measured by strain-gauge plethysmography, and pulmonary arterial pressure was measured by transthoracic echocardiography. Plasma nitrite doubled and clearance kinetics were similar whether nitrite was infused in hypoxia or normoxia. During hypoxia, nitrite increased forearm blood flow (+ 36%, P < 0.001) and reduced three separate indirect indexes of pulmonary arterial pressure by 16%, 12%, and 17% (P < 0.01). Pulmonary, but not systemic, arterial effects persisted 1 h after stopping the infusion, at a time when plasma nitrite had returned to baseline. No effects were observed during normoxia. Therefore, in hypoxic but not normoxic subjects, sodium nitrite causes arterial and pulmonary vasodilatation. In addition, hypoxia-induced pulmonary vasoconstriction was attenuated for a prolonged period and not dependent on a simultaneous elevation of plasma nitrite. This finding is consistent with the direct extravascular metabolism of nitrite to nitric oxide to effect hypoxia-associated bioactivity. [ABSTRACT FROM AUTHOR]

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 μm with L-nitroarginine reduced the [NO] ∼40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli. [ABSTRACT FROM AUTHOR]

Lund DD Chu Y, Miller JD, Heistad DD. Protective effect of extracellular superoxide dismutase on endothelial function during aging. Am J physiol Heart Circ Physiol 296: H1920-H1925, 2009. First published April 17, 2009; doi:10.1152/ajpheart.01342.2008.-Endothelial vasomotor function decreases with increasing age. Extracellular superoxide dismutase (ecSOD) protects against vascular dysfunction in several disease states. The purpose of this study was to determine whether endogenous ecSOD protects against endothelial dysfunction ip old mice. Vasomotor function of the aorta was studied ex vivo in wild-type (ecSOD[sup+/+]) and ecSOD-deficient (ecSOD[sup-/-]) mice at 11 (adult) and 29 (old) mo of age. Maximal relaxation to acetylcholine (10[sup-4] M) was impaired in vessels from adult ecSOD mice [75 ± (mean ± SE)1 compared with wild-type mice (89 ± 2%, P < 0.05). Maximal relaxation to acetylcholine (10[sup-4] M) was profoundly impaired in aorta from old ecSOD[sup-/-] mice (45 ± 5%) compared with wild-type mice (75 ± 4%, P < 0.05). There was a significant correlation between expression of ecSOD and maximal relaxation to acetylcholine in adult and old mice. Tempol (1 mM), a scavenger of uperoxide, improved relaxation in response to acetylcholine (63 8%) in old ecSOD[sup-/-] mice (P < 0.05), but not wild-type mice (75 ± 4%). Maximal relaxation to sodium nitroprusside was similar in aorta from adult and old wild-type and ecSOD[sup-/-] mice. Quantitative RT-PCR showed a decrease in mRNA levels of ecSOD and catalase in aorta of old mice and an increase in levels of TNFα and Nox-4 in aorta of old mice compared with adult mice. The findings support the hypothesis that impaired antioxidant mechanisms may contribute to cumulative increases in oxidative stress and impaired endothelial function in old mice. In conclusion, endogenous ecSOD plays an important role in protection against endothelial dysfunction during aging. [ABSTRACT FROM AUTHOR]

Peluffo G, Calcerrada P, Piacenza L, Pizzano N, Radi R. Superoxidemediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers. Am J P/iysiol Heart Circ Physiol 296: H 1781-H 1792, 2009. First published April 10, 2009; doi:10.1152/ajpheart.00930.2008.-Tobacco smoke is known to cause nitric oxide (∙NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between [sup∙]NO and superoxide (Or) radicals and the consequent impact on [sup&2219;]NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered [sub2][sup∙-] production as indicated by spin-trapping electron paramagnetic resonance experiments. 0 was produced both extracellulary (3.4 vs. 1.0 nmol∙h[sup-1]∙mg[sup-1] CSE vs. control; cytochrome c[sup3+] reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamme oxidation and protein tyrosine nitration on cells. O[sub2][sup∙-] and peroxynitrite formation were decreased by ascorbate and a-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-citocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert NO toward peroxynitrite by inducing O[sub2][sup∙-] production in vascular endothelial cells both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from ~50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 ± 241 vs. 1,059 ± 160 nM, P <0.01; and 26 ± 7 vs. 7 ± 1 µM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR. [ABSTRACT FROM AUTHOR]

Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction, and sudden death. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression, and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS-/- and CN/Tg mice were compared, some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS-/- mice had improved systolic performance (P < 0.001) and less heart block (P < 0.0001); larger sodium current density and lower serum TNF-α levels (P < 0.03); and less apoptosis (P < 0.01) resulting in improved survival (P < 0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS-/- mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, left ventricular dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin. [ABSTRACT FROM AUTHOR]

Hemoglobin-based oxygen carriers (HBOC) have been primarily studied for blood loss treatment. More recently infusions of HBOC in euvolemic subjects have been proposed for a wide variety of potential therapies in which increased tissue oxygenation would be beneficial. However, compared with the exchange transfusion models to study blood loss, less is known about HBOC oxygen delivery and vasoacitvity when it is infused in euvolemic subjects. We hypothesized that HBOC [polymerized bovine hemoglobin (PBvHb)] infusion creating hypervolemia would increase oxygen delivery to tissues during acute global hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused with 3 ml of either lactated Ringer or PBvHb solution (13 g/dl, 1.3 g/kg) during acute hypoxia (FIO2 = 10%, 4 h) or normoxia (FIO2 = 21%) exposure. Our data demonstrated that compared with Ringer-infused animals, in hypoxia and normoxia, PBvHb treatment improved oxygen content but raised mean arterial pressure, lowered stroke volume, heart rate, and cardiac index, which resulted in a net reduction in blood flow and oxygen delivery to the tissues. The PBvHb vasoactive effect was similar in magnitude and direction as to the Ringer-infused animals treated with a nitric oxide synthase inhibitor nitro-L-arginine, suggesting the PBvHb effect is mediated via nitric oxide scavenging. We conclude that infusion of PBvHb is not likely to be useful in treating global hypoxia under these conditions. [ABSTRACT FROM AUTHOR]

Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 µg/kg, subsequent dose 0.07 µg kg-1·day-1 im, 30 days) or vehicle. Blood levels at the end of treatment were 7.97 ± 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; 3) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM). Superoxide dismutase (SOD, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.3 mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, SOD did not affect phenylephrine responses; however, when coincubated with L-NAME, the L-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and SOD restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2- production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetyl- choline (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery (n = 50) under general anesthesia to induce MI or sham surgery (n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS (n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [³H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [³H]ACh release was significantly (P < 0.05) decreased in the Ml group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial {³H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days. [ABSTRACT FROM AUTHOR]

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS-1-) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1–10 Hz) and bath-applied norepinephrine (NE; 10-8–10-4 mol/l) on HR were examined in atria from nNOS-1- and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS-1- atria showed significantly reduced HR responses to sympathetic nerve activation and NE (P < 0.05). Similarly, the positive chronotropic response to the adenytate cyclase activator forskolin (10-7-10-5 mol/l) was attenuated in nNOS-1- atria (P < 0.05). Constitutive NOS inhibition with L-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS-1- and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes. [ABSTRACT FROM AUTHOR]

To learn whether nitric oxide (NO) inhalation can decrease myocardial ischemia-reperfusion (I/R) injury, we studied a murine model of myocardial infarction (MI). Anesthetized mice underwent left anterior descending coronary artery ligation for 30, 60, or 120 min followed by reperfusion. Mice breathed NO beginning 20 min before reperfusion and continuing thereafter for 24 h. MI size and area at risk were measured, and left ventricular (LV) function was evaluated using echocardiography and invasive hemodynamic measurements. Inhalation of 40 or 80 ppm, but not 20 ppm, NO decreased the ratio of MI size to area at risk. NO inhalation improved LV systolic function, as assessed by echocardiography 24 h after reperfusion, and systolic and diastolic function, as evaluated by hemodynamic measurements 72 h after reperfusion. Myocardial neutrophil infiltration was reduced in mice breathing NO, and neutrophil depletion prevented inhaled NO from reducing myocardial I/R injury. NO inhalation increased arterial nitrite levels but did not change myocardial cGMP levels. Breathing 40 or 80 ppm NO markedly and significantly decreased MI size and improved LV function after ischemia and reperfusion in mice. NO inhalation may represent a novel method to salvage myocardium at risk of UR injury. [ABSTRACT FROM AUTHOR]

Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for vascular endothelial growth factor-165 (Ad.VEGF-165) or endothelial nitric oxide (NO) synthase (Ad.eNOS) effectively augments skin viability in skin flap surgery and that the mechanism of Ad.VEGF-165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor NO. PBS (0.5 ml) or PBS containing Ad.VEGF-165, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally into the distal half of a mapped rat dorsal skin flap (4 × 10 cm) 7 days preoperatively, and skin flap viability was assessed 7 days postoperatively. Local subdermal gene therapy with 2 × 107-2 × 1010 plaque-forming units of VEGF-165 increased skin flap viability compared with PBS- or Ad.Null-injected control (P < 0.05). Subdermal Ad.VEGF-165 and Ad.eNOS gene therapies were equally effective in increasing skin flap viability at 5 × 108 plaque-forming units. Subdermal Ad.VEGF-165 therapy was associated with upregulation of eNOS protein expression, Ca2+-dependent NOS activity, synthesis/release of NO, and increase in capillary density and blood flow in the distal portion of the skin flap. Injection of the NOS inhibitor Nω-nitro-ʟ-arginine (15 mg/kg im), but not the cyclooxygenase inhibitor indomethacin (5 mg/kg im), 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by Ad.VEGF-165 injected subdermally into the mapped skin flap 7 days preoperatively, we have demonstrated for the first time that 1) Ad.VEGF-165 and Ad.eNOS mapped skin flap injected subdermally into the mapped skin flap 7 days preoperatively are equally effective in augmenting viability in the rat dorsal skin flap compared with control, 2) the mechanism of subdermal Ad.VEGF-165 gene therapy in augmenting skin flap viability involves an increase in NO synthesis/release downstream of upregulation of eNOS protein expression and Ca2+-dependent NOS activity, and 3) the vasodilating effect of NO may predominantly mediate subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability. [ABSTRACT FROM AUTHOR]

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations. [ABSTRACT FROM AUTHOR]

Brimonidine, an α2-adrenergic receptor (AR) agonist, has been employed in the treatment of glaucoma due to its beneficial effects on intraocular pressure reduction and neuroprotection. In addition, some studies have implicated that brimonidine might influence ocular blood flow; however, its effect on the retinal microcirculation has not been documented. Herein, we examined the vasomotor action of brimonidine on different branching orders of retinal arterioles in vitro and determined the contribution of the α2-AR subtype and the role of endothelium-derived nitric oxide (NO) in this vasomotor response. First- and second-order retinal arterioles of pigs were isolated, cannulated, and pressurized for functional studies. Video-microscopic techniques were employed to record diameter changes in response to brimonidine. RT-PCR was performed for detection of α-AR and endothelial NO synthase (eNOS) mRNA in retinal arterioles. All first-order arterioles (82 ± 2 μm ID) dilated dose dependently to brimonidine (0.1 nM to 10 μM) with 10% dilation at the highest concentration. Second-order arterioles (50 ± 1 μm ID) responded heterogeneously with either dilation or constriction. The incidence and magnitude of vasoconstriction were increased with increasing brimonidine concentration. Administration of the NO synthase inhibitor NG-nitro-ʟ-arginine methyl ester abolished the brimonidine-induced vasodilation in first- and second-order arterioles. Regardless of vessel size, vasomotor responses (i.e., vasodilation and vasoconstriction) of retinal arterioles were sensitive to the α2-AR antagonist rauwolscine. Consistent with the functional data, α2A-AR and eNOS mRNAs were detected in retinal arterioles. collectively, our data demonstrate that brimonidine at clinical doses evokes a consistent NO-dependent vasodilation in first-order retinal arterioles but a heterogeneous response in second-order arterioles. These vasomotor responses are mediated by the activation of α2-AR. It appears that brimonidine, depending on the concentration and vessel size, may alter local retinal blood flow. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10-6 to 10-4 M). Mouse MCAs, however, dilated by 21 ± 10% at 10-4 M CO. Authentic nitric oxide (NO·, 10-10 to 10-7 M) dilated both rat and mouse MCAs. At 10-8 M NO·, rat vessels dilated by 84 ± 4%, and at 10-7 M NO·, mouse vessels dilated by 59 ± 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor 8-aminolevulinic acid (10-10 to 10-4 M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 ± 2% at 3 × 10-5 M). Importantly, this constriction was unaltered by exogenous CO (10-4 M) or CO plus 10-5 M biliverdine (both HO products). In contrast, exogenous CO (10-4 M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 ± 1% in response to 10-5 M CrMP. Magnesium protoporphyrin IX (10-5 M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries. [ABSTRACT FROM AUTHOR]

The agonists of peroxisome proliferator-activated receptor-γ (PPARγ) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARγ agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARγ was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi (n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 ± 0.5 μM in N groups compared with 12.4 ± 0.6 μM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 ± 0.02 mm in the D group compared with 0.42 ± 0.01 mm in the N group. LV diastolic diameter was 3.05 ± 0.01 mm in the D group compared with 2.20 ± 0.02 mm in the N group. LV systolic diameter was 1.19 ± 0.02 mm in the D group and 0.59 ± 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARγ activity in high-fat, calorie-induced Type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow. [ABSTRACT FROM AUTHOR]

The effects of mechanical perturbations (shear stress, pressure) on microvascular permeability primarily have been examined in micropipette-cannulated vessels or in endothelial monolayers in vitro. The objective of this study is to determine whether acute changes in blood flow shear stress might influence measurements of hydraulic conductivity (Lp) in autoperfused microvessels in vivo. Rat mesenteric microvessels were observed via intravital microscopy. Occlusion of a third-order arteriole with a micropipette was used to divert and increase flow through a nonoccluded capillary or fourth-order arteriolar branch. Transvascular fluid filtration rate in the branching vessel was measured with a Landis technique. Flow (shear)-induced increases in Lp disappeared within 20-30 s of the removal of the shear and could be eliminated with nitric oxide synthase inhibition. The shear-induced increase in Lp was greater in capillaries compared with terminal arterioles. An acute change in shear may regulate Lp by a nitric oxide-dependent mechanism that displays heterogeneity within a microvascular network. [ABSTRACT FROM AUTHOR]

We examined the hypothesis that oxidants generated nitroso derivatives, activated latent matrix metalloproteinase (MMP), and induced proteinase-activated receptor 1 (PAR-I), leading to disconnection between the endothelium and myocytes. Administration of cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) ameliorated the oxidative-proteolytic stress and endothelial-myocyte uncoupling in chronic heart failure (CHF) in mice. Aortic-vena cava fistula (AVF) was created in 30 male mice (C57BL/6J) and studied at 0-, 2-, and 8-wk AVF. To reverse cardiac remodeling, as measured by MMP activation, purified CIMP was administered by an osmotic minipump subcutaneously after 8-wk AVF, and groups of mice (n = 6 mice/group) were examined after 12 and 16 wk. Levels of PAR-1 in the left ventricle (LV) were increased at 2 and 8 wk (compared with 0 wk of no CIMP treatment) but were normal at 12 and 16 wk after CIMP treatment, as measured by Western blot analysis. Similar results were obtained for LV levels of nitrotyrosine, MMP-2 and -9 activities, and TIMP-1 and -3. However, the levels of TIMP-4, endothelial cell density, and responses of cardiac rings to acetylcholine and bradykinin were attenuated at 2 and 8 wk and normalized after CIMP administration in AVF mice. CIMP induced nitric oxide in microvascular endocardial endothelial cells. The results suggest that nitro generation activated MMP and PAR-1, leading to endothelial-myocyte uncoupling. CIMP treatment normalized PAR-1 expression and ameliorated endothelial-myocyte uncoupling by decreasing oxidant-mediated proteolytic stress in CHF. [ABSTRACT FROM AUTHOR]

Protection against postinfarction myocardial dysfunction is modest with classic preconditioning (PC). We investigated whether multiple cycles of PC could improve this protection and whether postinfarction dysfunction only depends on the amount of viable tissue. Eighteen rabbits were chronically instrumented with coronary occluders and ultrasonic crystals (segment shortening, SH) in the ischemic zone. A control group underwent 30-min coronary artery occlusion (CAO) with 72-h reperfusion (CAR). In two other groups, PC was induced by six 4-min CAO/4-min CAR cycles (PC×6) or one 5-min CAO/10-min CAR cycle (PC×1). After 72-h CAR, depression in SH was reduced in PC×1 (-68 ± 7% from baseline) and to a greater extent in PC×6 (-18 ± 10%) vs. control (-99 ± 7%; all P < 0.05). Infarct sizes were reduced in PC×1 (15 ± 2%) and to a greater extent in PC×6 (3 ± 1%) vs. control (46 ± 5%; P < 0.05). Contractility of salvaged myocardium was evaluated by calculating the ratio between SH at 72-h CAR and the amount of viable tissue. This index was enhanced in PC×1 (0.39 ± 0.07, P < 0.05) and to a greater extent in PC×6 (0.82 ± 0.09) vs. control (0.0 ± 0.10). This differential effect of PC was not related to changes in apoptosis, endothelial nitric oxide synthase (NOS) expression, or macrophages infiltration but, rather, to blunted inducible NOS expression in PC×6 vs. control and PC×1. Thus multiple cycles of PC induced an almost complete protection against postinfarction dysfunction, potentially involving beneficial effects on salvaged myocardium. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the effects of topically applied triiodothyronine (T3) and thyroxine (T4) on the arterioles of hamster cheek pouch microcirculation in vivo. Microvessels were visualized using a fluorescent microscopy technique. Topical application of T3 (3.08, 30.8, 61.5, 307,615, and 6,150 nM/I) consistently induced dose-dependent dilation of arterioles within 2.0 ± 0.5 mm of administration. The application of Es (150, 257, 514, and 5,140 nM/l) caused different dose-dependent effects: dilation at the three lower doses within 16 ± 2 mm and rhythmic diameter changes at the highest dose. Aging of hamsters did not alter the arteriolar responses to T3 and T4. T3-induced dilation was countered by the inhibition of nitric oxide synthase with NG-nitro-L-arginine-methyl ester or NG-nitro-L-arginine. Iopanoic acid (IPA), which inhibits types I and II 5′-deiodinase, abolished the dilation elicited by 514 nM T4 but did not affect T3-dependent dilation. 6-Propyl-2-thiouracil (PTU), which inhibits type I 5′-deiodinase only, did not affect the dilation induced by T4. IPA and PTU did not impair arteriolar dilation induced by acetylcholine or sodium nitroprusside. These results indicate that T3 induces arteriolar dilation, likely through nitric oxide release. The local conversion of T4 to T3 appears to he crucial for the dilation induced by T4. [ABSTRACT FROM AUTHOR]

Hyperinsulinemia is a major risk factor for the development of vascular disease. We have reported that insulin increases the motility of vascular smooth muscle cells via hydrogen peroxide-mediated mechanism and that nitric oxide (NO) attenuates insulin-induced motility via a cGMP-mediated mechanism. Events downstream of cGMP elevation have not yet been investigated. The aim of our study was to test the hypothesis that antimotogenic effects of NO and cGMP in cultured rat aortic smooth muscle cells are mediated via PKG, followed by reduction of cytoplasmic Ca2+ levels and increased protein tyrosine phosphatase-proline, glutamate, serine, and threonine activity, leading to suppression of agonist-induced elevation of hydrogen peroxide levels and cell motility. Treatment of primary cultures with adenovirus expressing PKG-1α mimicked NO-induced inhibition of insulin-elicited hydrogen peroxide elevation and cell motility, whereas treatment with the pharmacological PKG inhibitor Rp-8-bromo-3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS) rescued the stimulatory effects of insulin that were suppressed by NO donor. Treatment of cells with insulin failed to increase cytoplasmic Ca2+ levels, whereas NO donor decreased cytoplasmic Ca2+ levels in the presence or absence of insulin. Treatment of cells with the Ca2+ chelator BAPTA mimicked the effects of PKG and the NO donor and increased the activity of PTP-PEST. Finally, treatment with a dominant negative allele of VIP-PEST reversed the inhibitory effect of BAPTA on cell motility and hydrogen peroxide elevation. We conclude that NO-induced inhibition of cell motility occurs via PKG-mediated reduction of basal cytoplasmic Ca2+ levels, followed by increased PTP-PEST activity, leading to decreased hydrogen peroxide levels and reduced cell motility. [ABSTRACT FROM AUTHOR]

This study compared the effects of rosuvastatin on left ventricular infarct size in mice after permanent coronary occlusion vs. 60 mm of ischemia followed by 24 h of reperfusion. Statins can inhibit neutrophil adhesion, increase nitric oxide synthase (NOS) expression, and mobilize progenitor stem cells after ischemic injury. Mice received blinded and randomized administration of rosuvastatin (20 mg·kg-1·day-1) or saline from 2 days before surgery until death. After 60 mm of ischemia with reperfusion, infarct size was reduced by 18% (P 0.03) in mice randomized to receive rosuvastatin (n = 18) vs. saline (n = 22) but was similar after permanent occlusion in rosuvastatin (n = 17) and saline (n = 20) groups (P = not significant). Myocardial infarct size after permanent left anterior descending coronary artery occlusion (n = 6) tended to be greater in NOS3-deficient mice than in the wild-type saline group (33 ± 4 vs. 23 ± 2%, P = 0.08). Infarct size in NOS3-deficient mice was not modified by treatment with rosuvastatin (34 ± 5%, n = 6, P = not significant vs. NOS3-deficient saline group). After 60 mm of ischemia-reperfusion, neutrophil infiltration was similar in rosuvastatin and saline groups as was the percentage of CD34+, Sca-1, and c-Kit+ cells. Left ventricular NOS3 mRNA and protein levels were unchanged by rosuvastatin. Rosuvastatin reduces infarct size after 60 mm of ischemia-reperfusion but not after permanent coronary occlusion, suggesting a potential anti-inflammatory effect. Although we were unable to demonstrate that the myocardial protection was due to an effect on neutrophil infiltration, stem cell mobilization, or induction of NOS3, these data suggest that rosuvastatin may be particularly beneficial in myocardial protection after ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

To study the mechanisms of vascular dysfunction in diabetes mellitus, we examined the responses of the aorta to adrenomedullin (AM) and ANG II in obese Zucker (OZ), lean Zucker (LZ), and OZ rats administered fluvastatin (OZ + Flu). AM-induced endothelium-dependent vasorelaxation was impaired in OZ rats compared with LZ rats, and fluvastatin restored AM-induced, endothehum-dependent vasorelaxation (%Δtension at 10-7 molfi AM; LZ, -85.1 ± 3.1%; OZ, -50.7 ± 2.5%; OZ + Flu, -75.6 ± 2.7%). Expression of endothelial nitric oxide synthase (eNOS) and Akt phosphorylation in response to AM (10-7 mol/l) were also diminished in OZ rats. Fluvastatin restored the eNOS expression and Akt phosphorylation [eNOS expression (relative intensity); LZ, 2.3 ± 0.4; OZ, 1.0 ± 0.2; OZ + Flu, 1.8 ± 0.3; Akt phosphorylation (relative intensity); LZ, 2.3 ± 0.2; OZ, 1.0 ± 0.3; OZ + Ru, 1.9 ± 0.2]. ANG II-induced vasoconstriction was enhanced in the aortic rings of OZ rats compared with LZ rats, and this enhanced vasoconstriction was partially normalized by fluvastatin and was abolished when the aorta of OZ rats was preincubated with the Rho kinase inhibitor Y-27632. GTPγS-induced contraction of permeabilized aortic smooth muscle cells, which is an indicator of the Rho-dependent Ca2+ sensitization of contraction, was enhanced in OZ rats compared with LZ rats, and this enhanced contraction was suppressed in OZ + Flu rats. These results suggested that endothelium-dependent vasorelaxation was impaired, Ca2+ sensitization of contraction was augmented in blood vessels of OZ rats and that fluvastatin restored vascular function by activating the Akt-dependent pathway and inhibiting the Rho-dependent pathway. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K+ channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K+ channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nunprotein contaminant. Sodium azide (NaN3, 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN3 elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K+ channels. NaN3-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone, Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN3 and NaN3-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN3 (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone. [ABSTRACT FROM AUTHOR]

Elevations in arterial pressure associated with hypertension, microgravity, and prolonged bed rest alter cerebrovascular autoregulation in humans. Using head-down tail suspension (HDT) in rats to induce cephalic fluid shifts and elevate arterial pressure, this study tested the hypothesis that 2-wk HDT enhances cerebral artery vasoconstriction and that an enhanced vasoconstriction described in vitro will alter regional cerebral blood flow (CBF) and vascular resistance (CVR) during standing and head-up tilt. To test this hypothesis, basal tone and vasoconstrictor responses to increases in transmural pressure, shear stress, and K+ were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME; l0-5 M) and with endothelium removal. Endothelial NOS (eNOS) mRNA and protein expression levels were measured by RT-PCR and immunoblot, respectively. Regional CBF and CVR were determined with a radiolabeled tracer technique and quantitative autoradiography. Basal tone and all vasoconstrictor responses were greater in MCAs from KDT rats. L-NAME and endothelium removal abolished these differences between groups, and HDT was associated with lower levels of MCA eNOS protein. CBF in select regions was lower and CVR higher during standing and head-up tilt in HDT rats. These results indicate that chronic cephalic fluid shifts enhanced basal tone and vasoconstriction through alterations in the eNOS signaling mechanism. The functional consequence of these vascular alterations with HDT is regional elevations in CVR and corresponding reductions in cerebral perfusion. [ABSTRACT FROM AUTHOR]

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice (P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of I-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel­Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel­Palade body contents (and 1-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel­Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency. [ABSTRACT FROM AUTHOR]

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K+ (mitoKATP) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 mm of anoxia followed by 60 mm of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2′ ,7′ -dichlorofluorescin (DCFH). Effects of the specific mitoKATP channel opener diazoxide (Diazo, 50 μM) or the blocker 5-hydroxydecanoate (5-HD. 500 μM), the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 50 νM), the antioxidant N-(2-mercaptopropionyl) glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 μM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 mm of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-RD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoKATP channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium. [ABSTRACT FROM AUTHOR]

Erythrocytes (red blood cells) of either rabbits or healthy humans are required to demonstrate the participation of nitric oxide (NO) in the regulation of pulmonary vascular resistance in the isolated rabbit lung. The property of the erythrocyte that is responsible for the stimulation of NO synthesis was reported to be the ability to release ATP in response to physiological stimuli, including deformation. Moreover, a signal transduction pathway that relates mechanical deformation of erythrocytes to ATP release has been described, and the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a component, i.e., erythrocytes of individuals with CF do not release ATP in response to deformation. Here, we investigated the hypothesis that, in contrast to those of healthy humans, erythrocytes of humans with CF fail to stimulate endogenous NO synthesis in the isolated rabbit lung. We report that CFTR is a component of the membranes of both rabbit and human erythrocytes. The addition of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM) produced increases in vascular resistance in isolated rabbit lungs perfused with physiological salt solution (P55) containing erythrocytes of healthy humans, but L-NAME was without effect when the lungs were perfused with P55 alone or PSS containing erythrocytes of CF patients. These results provide support for the hypothesis that, in CF, a defect in ATP release from erythrocytes could lead to decreased endogenous pulmonary NO synthesis and contribute to pulmonary hypertension. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the time course of flow-induced vasodilation in soleus and gastrocnemius muscle arterioles and the mechanisms that underlie vasodilatory responses to an increase in intraluminal flow. Vasodilation was assessed during 20 mm of continuous exposure to intraluminal flow. Both soleus and gastrocnemius muscle arterioles dilated in response to flow, although the magnitude of vasodilation was greater in arterioles from the gastrocnemius muscle. Neither blockade of nitric oxide synthase with NG-nitro-L-arginine methyl ester (L-NAME) nor blockade of cyclooxygenase with indomethacin inhibited the initial vasodilation (0–2 min) in arterioles from either muscle. In contrast, vasodilation to sustained exposure to flow (2–20 min) was eliminated by treatment with L-NAME in arterioles from both muscles. Both depolarization with 40 mM KC1 and blockade of Ca2+-activated K+ channels inhibited the initial flow-induced dilation, and the inhibition was greater in gastrocnemius muscle arterioles than soleus muscle arterioles, In the presence of L-NAME, prolonged exposure to flow resulted in constriction in soleus and gastrocnemius muscle arterioles. This constriction was abolished by endothelin receptor blockade. These results indicate that the time course and magnitude of flow-induced vasodilation differs between arterioles from soleus and gastrocnemius muscles. The immediate response to increased flow is greater in gastrocnemius muscle arterioles and involves activation of K+ channels. In arterioles from both soleus and gastrocnemius muscles, vasodilation to sustained flow exposure occurs primarily through production of nitric oxide. In the absence of nitric oxide, sustained exposure to flow results in pronounced constriction that is mediated by endothelin. [ABSTRACT FROM AUTHOR]

Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a poweful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg iv each) 24 h before 30 min of coronary artery occlusion followed by 24 h of repeffusion. Infarct size was significantly reduced in DETA/NO-treated ngne (30 ± 2% of risk region in treated mice vs 50 ± 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg iv), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 ± 1 vs, 30 ± 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca2+-thdueed swelling by 20 μmol/l CaCl2 or by the higher concentration of 200 μmol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 μmol/l CaCl2, thereby confirming that Ca2+ induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca2+-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator. [ABSTRACT FROM AUTHOR]

Inducible nitric oxide synthase (iNOS) has been implicated in the pathophysiology of congestive heart failure (CHF). Given the extensive evidence supporting this concept, we hypothesized that iNOS deficiency (iNOS-/-) would attenuate the severity of CHF in mice. Mice were subjected to permanent occlusion [myocardial infarction (MI)] of the proximal left anterior descending coronary artery to produce CHF. Cardiac function was assessed in vivo using echocardiography and ultraminiature ventricular pressure catheters. Sham wild-type (n = 17), sham iNOS-/- (n=8), Ml wild type (n = 56), andMI iNOS-/- (n = 48) mice were subjected to MI (or sham MI) and followed for 1 mo. Deficiency of iNOS did not alter survival during CHF compared with wild type (35% vs. 32%, P = not significant). Furthermore, fractional shortening and cardiac output were not significantly different between wild-type (9.6 ± 2.0% and 441 ± 20 μl·min-1·g-1) and iNOS -/- (9.8 ± 1.3% and 471 ± 26 μl·min-1·g -1) mice. The extent of cardiac hypertrophy and pulmonary edema was also similar between wild-type and iNOS-/- mice. None of the indexes demonstrated any significant differences between iNOS-/- and wild-type mice sub- jected to MI. These findings indicate that deficiency of iNOS does not significantly affect severe CHF in mice after M1. [ABSTRACT FROM AUTHOR]

The effects of extracellular K[sup +] on endothelium-dependent relaxation (EDR) and on intracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F[sub 2α] or norepinephrine, an increase in extracellular K[sup +] concentration ([K[sup +]][sub o]) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K[sup +]][sub o] inhibited the agonist-induced [Ca[sup 2+]][sub i] increase concentration dependently. Similar to K[sup +], Cs[sup +] also inhibited EDR and the increase in [Ca[sup 2+]][sub i] concentration dependently. In current-clamped HUVEC, increasing [K[sup +]][sub o] from 6 to 12 mM depolarized membrane potential from -32.8 ± 2.7 to -8.6 ± 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca[sup 2+]][sub i] significantly from 0.95 ± 0.03 to 0.88 ± 0.03 µM (n = 11, P < 0.01) and further decreased [Ca[sup 2+]][sub i] to 0.47 ± 0.04 µM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATPinduced [Ca[sup 2+][sub i] increase in voltage-clamped MAEC. The intermediate-conductance Ca[sup 2+]-activated K[sup +] channel openers 1-ethyl-2benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K[sup +]-induced inhibition of EDR and increase in [Ca[sup 2+]][sub i]. The K[sup +]-induced inhibition of EDR and increase in [Ca[sup 2+]][sub i] was abolished by the Na[sup +]-K[sup +] pump inhibitor ouabain (10 µM). These results indicate that an increase of [K[sup +]][sub o] in the physiological range (6-12 mM) inhibits [Ca[sup 2+]][sub i] increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K[sup +] efflux, and activating the Na[sup +]-K[sup +] pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone. [ABSTRACT FROM AUTHOR]

The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO·) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO· exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO' production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO· (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration. [ABSTRACT FROM AUTHOR]

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca[sup 2+] and inhibited by blocking electrogenic Ca[sup 2+] uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca[sup 2+], increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting, mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO[sup -]) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the significance of two intracellular scavengers of nitric oxide (NO): 1) superoxide dismutase (SOD) (SOD2) to scavenge intramitochondrial superoxide anion, and 2) cytosolic myoglobin (Mb) in the regulation of tissue O[sub 2] consumption. 02 consumption was measured in vitro using a Clark-type O[sub 2] electrode. SOD heterozygous mice (SODHZ) (n = 13) and SOD wildtype (SODWT) (n = 5) mice were used. Bradykinin (BK, 10[sup -4] mol/l) reduced O[sub 2] consumption by 15% ± 1 in hearts of SODHZ mice, which was significantly different from SODWT (reduced by 24 ± 0.4%). Tiron significantly increased the inhibition of O[sub 2] consumption by BK in male mice from 15 ± 1% (n = 13) to 29 ± 1.2% (n = 4) at 10[sup -4] mo1/l concentration (P < 0.05). The effect of carbachol was similar to BK. S-nitroso-N-acetyl penicillamine (SNAP, 10[sup -4] mol/l) reduced O[sub 2] consumption by 39 ± 1.3% in hearts of SODHZ mice, which was not significantly different from SODWT. But at 10[sup -7] mol/l, SNAP caused significantly less inhibition of O[sub 2] consumption in SODHZ mice. Mb knockout (MbKO; Mb wild-type n = 6) and (MbWT) mice (n = 6) were also used. Kidney cortex was studied as the negative control because it does not contain Mb. BK (10[sup -4] mol/l) reduced O[sub 2] consumption by 32 ± 2, 29 ± 1, and 26 ± 1% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. SNAP (10[sup -]4 mol/l) reduced O[sub 2] consumption by 39 ± 3, 42 ± 4, and 46 ± 2% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. N[sup G]-nitro-L-arginine methyl ester (P < 0.05) inhibited the reduction in O[sub 2] consumption induced by BK in the MbKO mouse heart (15 ± 1%), skeletal muscle (17 ± 1%), and kidney (17 ± 1%) as in the MbWT mice. These results suggest that the role of Mb as an intracellular NO scavenger is small, and the increase in mitochondrial superoxide in SODHZ mice may cause a decrease NO bioavailability and alter the control of myocardial O[sub 2] consumption by NO. [ABSTRACT FROM AUTHOR]

Investigates the effects of cardiac pacing on the functions of nitric oxide and forearm vascular responses. Increase blood flow; Association of pulse frequency with flow-mediated dilatation; Attenuation of the pulse frequency with detector vessel dilatation.

Discusses a study which examined the mechanism by which Guanosine 3',5'-cyclic Monophosphate (cGMP) contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries. Details of animal experiments conducted; Effects of NO and cGMP on the vasoconstrictor response to calcium; Results; Discussion.

Presents a study that found that low-volume anoxia and reoxygenation increased nitric oxide synthase (NOS) activity and facilitated the conversion of L-arginine to nitric oxide (NO) to protect against heart cell injury. Measurement of NO production; Characterization of NOS isoforms in human heart cells after low-volume anoxia and reoxygenation; Effects of L-arginine on cellular injury and NO production after low-volume anoxia and reoxygenation.

Presents a study that investigated the effect of nitric oxide on endothelium-derived hyperpolarizing factor (EDHF) response in the middle cerebral arteries of rats. Background on the mechanism of the EDHF response; Methods used; Results and discussion.

Focuses on a study that examined if nitric oxide (NO) contributes to norepinephrine-induced right coronary vasolidation and oxygen consumption. Background on the effect of NO on vascular smooth muscles; Methods used; Results and discussion.

Presents a study that assessed the contribution of nitric oxide formation to exercise-induced coronary responses after α-adrenergic receptor blockade. Background on the balance between coronary blood flow and myocardial oxygen consumption during exercise; Methods used; Results and discussion.

Focuses on a study which examined the effect of increased release of nitric oxide by nitric oxide synthase can contribute to the increased blood flow to calf muscles after acute femoral artery occlusion. Methodology of the study; Results and discussion.

Presents a study which examined whether heat shock protein 90, phosphatidylinositol-3-kinase, and tyrosine kinase participated in agonist-induced endothelial nitric oxide synthase activation in cerebral arterioles in vivo. Methodology of the study; Results and discussion.

Focuses on a study which investigated whether geldanamycin can attenuate nitric oxide-mediated dilation in human skin. Methodology of the study; Results and discussion.

Presents a study which investigated the effect of nitric oxide in inducing apoptosis by activating potassium channels in pulmonary vascular smooth muscle cells. Methodology and materials; Results and discussion.

Presents information on a study which investigated the role of nitric oxide in exercise-induced arteriogenesis and angiogenesis. Background on arteriogenesis and angiogenesis; Methodology; Results of the study.