Basal Endothelial Nitric Oxide Research Articles

Shear stress-induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure.

Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.

Nebivolol and Endothelial Dysfunction in Patients With Essential Hypertension: A Reputation Saver of β‐Blockers?

We have read with great interest the recently published article by Neuman and coworkers1 in which they hypothesized that nebivolol, a selective β1-antagonist that stimulates nitric oxide (NO), improves endothelial function in African Americans with hypertension when compared with metoprolol. In that well-designed study, the authors found evidence for NO bioavailability at rest during treatment with both nebivolol and metoprolol succinate in hypertensive African American patients, with a clear trend for a greater contribution of NO during nebivolol therapy. Moreover, after combined blockade of NO and endothelium-derived hyperpolarizing factor (EDHF), there was significantly greater vasoconstriction during nebivolol compared with metoprolol therapy, suggesting a greater contribution of both NO and EDHF combined to resting vasomotor tone during nebivolol treatment. Moreover, the contribution of NO to exercise-induced vasodilation was greater during treatment with nebivolol compared with metoprolol succinate. Nebivolol is a third-generation, β1-adrenergic receptor antagonist that has vasodilatory properties both in animals and humans independent of β1-receptor antagonism and related to β3-receptor agonist effects.2-5 It is a racemic mixture of D- and L-enantiomers, of which D-nebivolol is considered to be a highly selective β-adrenergic receptor antagonist.5-8 It was demonstrated that nebivolol stimulates NO release through both β3-receptor and adenosine triphosphate–dependent, P2Y-receptor activation.3, 9-12 In a well-designed clinical study, Tzemos and coworkers13 analyzed whether nebivolol could improve endothelial dysfunction in patients with essential hypertension. They showed that nebivolol/bendrofluazide increased both stimulated and basal endothelial NO release, whereas, for the same degree of blood pressure control, atenolol/bendrofluazide had no effect on NO bioactivity; therefore, they concluded that nebivolol may offer additional vascular protection in treating hypertension.13 Moreover, Oelze and coworkers14 demonstrated that nebivolol but not metoprolol improved endothelial function and reduced vascular oxidative stress in an experimental model of AngII-induced hypertension. These effects were associated with a normalization of the expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits Nox1, Nox2, p22phox, p47phox, p67phox, and Rac1 and an inhibition of NOS III uncoupling in a rat hypertension model. More importantly, they found that nebivolol but not metoprolol inhibited activation of vascular NADPH oxidase and were able to dissociate an already assembled and active membrane-associated NADPH oxidase complex. These observations indicate that nebivolol, in addition to its β1-receptor–blocking and NO-releasing effects, possesses substantial inhibitory effects on vascular oxidative stress, all of which may beneficially influence endothelial dysfunction because of oxidative stress in the setting of arterial hypertension.14 It is well-known that essential hypertension is associated with endothelial dysfunction, and with greater oxidative stress producing free oxygen radicals that contribute to the decrease in NO bioavailibility. Elevated plasma levels of malondialdehyde, a final product of lipid peroxidation and an index of oxidative stress, have been demonstrated in patients with essential hypertension.15, 16 Concordantly, our study group previously found that nebivolol, differently from metoprolol, improved oxidative stress and insulin sensitivity, decreased plasma sP-selectin, and increased adiponectin levels in hypertensive patients.17 From that standpoint, we concluded that nebivolol may significantly improve endothelial dysfunction in hypertensive patients. However, our study population had no African American patients, unlike the patient population from the study by Neumann and coworkers. In conclusion, nebivolol has beneficial effects on endothelial function mainly by increasing NO bioavailability. We strongly believe that although standard β-blockers are falling out of favor nowadays, perhaps the new generation of β-blockers such as nebivolol may “rescue” the reputation of these agents in the near future. There are no conflicts of interest to declare.

Pial Collateral Reactivity During Hypertension and Aging: Understanding the Function of Collaterals for Stroke Therapy.

We investigated vasoactive properties of leptomeningeal arterioles (LMAs) under normotensive conditions and during hypertension and aging that are known to have poor collateral flow and little salvageable tissue. LMAs, identified as distal anastomotic arterioles connecting middle and anterior cerebral arteries, were studied isolated and pressurized from young (18 weeks) or aged (48 weeks) normotensive Wistar Kyoto (WKY18, n=14; WKY48, n=6) rats and spontaneously hypertensive rats (SHR18, n=16; SHR48, n=6). Myogenic tone and vasoactive responses to pressure as well as endothelial function and ion channel activity were measured. LMAs from WKY18 had little myogenic tone at 40 mm Hg (8±3%) that increased in aged WKY48 (30±6%). However, LMAs from both WKY groups dilated to increased pressure and demonstrated little myogenic reactivity, a response that would be conducive to collateral flow. In contrast, LMAs from both SHR18 and SHR48 displayed considerable myogenic tone (56±8% and 43±7%; P<0.01 versus WKY) and constricted to increased pressure. LMAs from both WKY and SHR groups had similar basal endothelial nitric oxide and IK channel activity that opposed tone. However, dilation to sodium nitroprusside, diltiazem and 15 mmol/L KCl was impaired in LMAs from SHR18. This study shows for the first time that LMAs from young and aged SHR are vasoconstricted and have impaired vasodilatory responses that may contribute to greater perfusion deficit and little penumbral tissue. These results also suggest that therapeutic opening of pial collaterals is possible during middle cerebral artery occlusion to create penumbral tissue and prevent infarct expansion.

CRP/oxLDL co-incubates impair endothelial functions through CD32, LOX-1, and keratin 1 with dependence on their ratio

C-reactive protein (CRP) and oxidized low-density lipoprotein (oxLDL) play a significant role in the pro-inflammatory response observed in various vascular diseases. CRP has a characteristic cyclic pentameric structure that forms two binding sites known as the A and B faces. The CRP B face can bind five oxidized phosphatidylcholine molecules in oxLDL in the presence of Ca2+. However, how CRP/oxLDL complexes are formed, as well as their effect and corresponding receptors on endothelial cells, remains unknown. In the present study, we found that the inhibition of basal endothelial nitric oxide (NO) release by CRP and oxLDL co-incubates is dependent on the ratio of CRP and oxLDL. Specifically, the concentration ratio of CRP to oxLDL ∼1:20 (CRP/oxLDL-1:20) results in the greatest inhibition of endothelial NO secretion. Pretreatment with neutralizing antibodies for CD32, the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), or keratin 1 (KRT1) abolished the effect of CRP/oxLDL-1:20 on NO production. Furthermore, we found that CD32, LOX-1, and KRT1 mediate the cellular uptake of CRP/oxLDL complexes, in which LOX-1 co-localizes with KRT1, but not CD32. These results provide a novel mechanism by which the multifaceted effects of CRP/oxLDL complexes on endothelial cells can be targeted for the treatment of vasculature diseases.

P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial mechanotransduction.

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress-induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y2 and the G proteins Gq/G11 mediate fluid shear stress-induced endothelial responses, including [Ca2+]i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y2 receptor. Mice with induced endothelium-specific P2Y2 or Gq/G11 deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y2 and Gq/G11 as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone, and blood pressure.

Protein Kinase C-β Contributes to Impaired Endothelial Insulin Signaling in Humans With Diabetes Mellitus

Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling, through the activity of protein kinase C-β (PKCβ) and nuclear factor κB, reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. We measured protein expression and insulin response in freshly isolated endothelial cells from patients with type 2 diabetes mellitus (n=40) and nondiabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes mellitus (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in nondiabetic subjects but not in diabetic patients (P=0.003), consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients, indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=-0.541, P=0.02). Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor κB activation was higher in diabetes mellitus and was reduced with PKCβ inhibition. We provide evidence for the presence of altered eNOS activation, reduced insulin action, and inflammatory activation in the endothelium of patients with diabetes mellitus. Our findings implicate PKCβ activity in endothelial insulin resistance.

Age-Dependent Rescue by Simvastatin of Alzheimer's Disease Cerebrovascular and Memory Deficits

Alzheimer's disease (AD) is now established as a progressive compromise not only of the neurons but also of the cerebral vasculature. Increasing evidence also indicates that cerebrovascular dysfunction may be a key or an aggravating pathogenic factor in AD, emphasizing the importance to properly control this deficit when aiming for effective therapy. Here, we report that simvastatin (3-6 months, 40 mg/kg/d) completely rescued cerebrovascular reactivity, basal endothelial nitric oxide synthesis, and activity-induced neurometabolic and neurovascular coupling in adult (6 months) and aged (12 months) transgenic mice overexpressing the Swedish and Indiana mutations of the human amyloid precursor protein (AD mice). Remarkably, simvastatin fully restored short- and long-term memory in adult, but not in aged AD mice. These beneficial effects occurred without any decreasing effect of simvastatin on brain amyloid-β (Aβ) levels or plaque load. However, in AD mice with recovered memory, protein levels of the learning- and memory-related immediate early genes c-Fos and Egr-1 were normalized or upregulated in hippocampal CA1 neurons, indicative of restored neuronal function. In contrast, the levels of phospholipase A2, enkephalin, PSD-95, synaptophysin, or glutamate NMDA receptor subunit type 2B were either unaltered in AD mice or unaffected by treatment. These findings disclose new sites of action for statins against Aβ-induced neuronal and cerebrovascular deficits that could be predictive of therapeutic benefit in AD patients. They further indicate that simvastatin and, possibly, other brain penetrant statins bear high therapeutic promise in early AD and in patients with vascular diseases who are at risk of developing AD.

Endothelial function in migraine: a cross-sectional study

BackgroundMigraine has been associated with cardiovascular disorders. Endothelial dysfunction may be a mechanism underlying this association. The present study tested the hypothesis that endothelium-dependent vasodilation, basal endothelial nitric oxide release and endothelial fibrinolytic capacity are impaired in migraine patients.MethodsGraded doses of sodium nitroprusside (SNP, 0.2 to 0.8 μg.min-1.dL-1 forearm), substance P (0.2 to 0.8 pmol.min-1.dL-1 forearm) and NG-monomethyl-L-arginine (L-NMMA, 0.1 to 0.4 μmol.min-1.dL-1 forearm) were infused into the brachial artery of 16 migraine patients with or without aura during a headache-free interval and 16 age- and sex-matched subjects without a history of migraine. Forearm blood flow (FBF) was measured by strain-gauge venous occlusion plethysmography. Local forearm release of tissue plasminogen activator (t-PA) in response to substance P infusion was assessed using the arteriovenous plasma concentration gradient. Responses to infused drugs were compared between patients and matched controls by analysis of variance.ResultsIn both migraine patients and control subjects, SNP and substance P caused a dose-dependent increase, and L-NMMA a dose-dependent decrease in FBF (P < 0.001 for all responses). In both groups, substance P caused an increase in t-PA release (P < 0.001). FBF responses and t-PA release were comparable between migraine patients and control subjects.ConclusionsThe absence of differences in endothelium-dependent vasodilation, basal endothelial nitric oxide production and stimulated t-PA release between migraine patients and healthy control subjects argues against the presence of endothelial dysfunction in forearm resistance vessels of migraine patients.

Knockout of insulin receptors in cardiomyocytes attenuates coronary arterial dysfunction induced by pressure overload

Ablating insulin receptors in cardiomyocytes causes subendocardial fibrosis and left ventricular (LV) dysfunction after 4 wk of transverse aortic constriction (TAC). To determine whether these maladaptive responses are precipitated by coronary vascular dysfunction, we studied mice with cardiomyocyte-restricted knock out of insulin receptors (CIRKO) and wild-type (WT) TAC mice before the onset of overt LV dysfunction. Two weeks of TAC produced comparable increases (P < 0.05 vs. respective sham) in heart weight/body weight (mg/g) in WT-TAC (8.03 ± 1.14, P < 0.05 vs. respective sham) and CIRKO-TAC (7.76 ± 1.25, P < 0.05 vs. respective sham) vs. WT-sham (5.64 ± 0.11) and CIRKO-sham (4.64 ± 0.10) mice. In addition, 2 wk of TAC were associated with similar LV geometry and function (echocardiography) and interstitial fibrosis (picrosirius red staining) in CIRKO and WT mice. Responses to acetylcholine (ACh), N(G)-monomethyl-L-arginine (l-NMMA), and sodium nitroprusside (SNP) were measured in coronary arteries that were precontracted to achieve ∼70% of maximal tension development using the thromboxane A(2) receptor mimetic U-46619 (∼3 × 10(-6) M). ACh-evoked vasorelaxation was absent in WT-TAC but was present in CIRKO-TAC albeit reduced relative to sham-operated animals. l-NMMA-evoked tension development was similar in vessels from CIRKO-TAC mice but was lower (P < 0.05) in WT-TAC animals vs. the respective sham-operated groups, and SNP-evoked vasorelaxation was similar among all mice. Thus estimates of stimulated and basal endothelial nitric oxide release were better preserved in CIRKO vs. WT mice in response to 2 wk of TAC. These findings indicate that maladaptive LV remodeling previously observed in CIRKO-TAC mice is not precipitated by coronary artery dysfunction, because CIRKO mice exhibit compensatory mechanisms (e.g., increased eNOS transcript and protein) to maintain coronary endothelial function in the setting of pressure overload.

Nitric oxide and superoxide transport in a cross section of the rat outer medulla. II. Reciprocal interactions and tubulovascular cross talk

In a companion study (Edwards A and Layton AT. Am J Physiol Renal Physiol. doi:10.1152/ajprenal.00680.2009), we developed a mathematical model of nitric oxide (NO), superoxide (O(2)(-)), and total peroxynitrite (ONOO) transport in mid-outer stripe and mid-inner stripe cross sections of the rat outer medulla (OM). We examined how the three-dimensional architecture of the rat OM, together with low medullary oxygen tension (Po(2)), affects the distribution of NO, O(2)(-), and ONOO in the rat OM. In the current study, we sought to determine generation rate and permeability values that are compatible with measurements of medullary NO concentrations and to assess the importance of tubulovascular cross talk and NO-O(2)(-) interactions under physiological conditions. Our results suggest that the main determinants of NO concentrations in the rat OM are the rate of vascular and tubular NO synthesis under hypoxic conditions, and the red blood cell (RBC) permeability to NO (P(NO)(RBC)). The lower the P(NO)(RBC), the lower the amount of NO that is scavenged by hemoglobin species, and the higher the extra-erythrocyte NO concentrations. In addition, our results indicate that basal endothelial NO production acts to significantly limit NaCl reabsorption across medullary thick ascending limbs and to sustain medullary perfusion, whereas basal epithelial NO production has a smaller impact on NaCl transport and a negligible effect on vascular tone. Our model also predicts that O(2)(-) consumption by NO significantly reduces medullary O(2)(-) concentrations, but that O(2)(-) , when present at subnanomolar concentrations, has a small impact on medullary NO bioavailability.

Vascular Inflammation in Obesity and Sleep Apnea

Unrecognized obstructive sleep apnea (OSA) is highly prevalent in obesity. Both obesity and OSA are associated with vascular endothelial inflammation and increased risk for cardiovascular diseases. We investigated directly whether the endothelial alterations that are attributed commonly to obesity are in fact related to OSA. Seventy-one subjects with a body mass index ranging from normal to obese underwent attended polysomnography. To assess vascular inflammation and oxidative stress directly, we quantified the expression of nuclear factor-kappaB and nitrotyrosine by immunofluorescence in freshly harvested venous endothelial cells. To evaluate basal endothelial nitric oxide (NO) production and activity, we quantified the expression of endothelial NO synthase (eNOS) and phosphorylated eNOS. Vascular reactivity was measured by brachial artery flow-mediated dilation. Expression of eNOS and phosphorylated eNOS and flow-mediated dilation were significantly lower, whereas expression of nitrotyrosine was significantly greater in OSA patients (n=38) than in OSA-free subjects (n=33) regardless of central adiposity. Expression of nuclear factor-kappaB was greater in obese OSA patients than in obese OSA-free subjects (P=0.004). Protein expression and flow-mediated dilation were not significantly affected by increasing body mass index or central obesity in OSA patients and in OSA-free subjects. After 4 weeks of continuous positive airway pressure therapy, flow-mediated dilation and expression of eNOS and phosphorylated eNOS significantly increased whereas expression of nitrotyrosine and nuclear factor-kappaB significantly decreased in OSA patients who adhered to continuous positive airway pressure >/=4 hours daily. Untreated OSA rather than obesity is a major determinant of vascular endothelial dysfunction, inflammation, and elevated oxidative stress in obese patients.

Basal endothelial nitric oxide synthase (eNOS) phosphorylation on Ser 1177 occurs in a stable microtubule- and tubulin acetylation-dependent manner

To better understand the relationship between the subcellular compartmentalization of endothelial nitric oxide synthase (eNOS) and its function in endothelial cells, we addressed the roles of the microtubule network and of its dynamics in organizing Golgi-bound eNOS. We found that part of Golgi-bound eNOS localizes to the trans-Golgi network and/or to trans-Golgi network-derived vesicles and membrane tubules that are organized preferentially by stable microtubules. Also, while most of cellular eNOS was recovered in a detergent-resistant microtubule-enriched subcellular fraction, its recovery was impaired after total microtubule disassembly, but not after selective disassembly of dynamic microtubules or after microtubule stabilization. Basal eNOS phosphorylation on Ser 1177 further required the association of the trans-Golgi network to stable microtubules and was enhanced by microtubule stabilization. We finally show that the involvement of stable microtubules in basal eNOS phosphorylation involved alpha-tubulin acetylation. Microtubule-dependent organization of subcellular eNOS and control over its phosphorylation would thus be essential for endothelial cells to maintain their basal eNOS function.

Tacrolimus reduces nitric oxide synthase function by binding to FKBP rather than by its calcineurin effect

Hypertension develops in many patients receiving the immunosuppressive drug tacrolimus (FK506). One possible mechanism for hypertension is a reduction in vasodilatory nitric oxide. We found that tacrolimus and a calcineurin autoinhibitory peptide significantly decreased vascular calcineurin activity; however, only tacrolimus altered intracellular calcium release in mouse aortic endothelial cells. In mouse aortas, incubation with tacrolimus increased protein kinase C activity and basal endothelial nitric oxide synthase phosphorylation at threonine 495 but reduced basal and agonist-induced endothelial nitric oxide synthase phosphorylation at serine 1177, a mechanism known to inhibit synthase activity. While this decreased nitric oxide production and endothelial function, the calcineurin autoinhibitory peptide had no such effects. Inhibition of ryanodine receptor opening or protein kinase C blocked the effects of tacrolimus. Since it is known that the FK506 binding protein (FKBP12/12.6) interacts with the ryanodine receptor to regulate calcium release, we propose this as the mechanism by which tacrolimus alters intracellular calcium and endothelial nitric oxide synthase rather than by its effect on calcineurin. Our study shows that prevention of the tacrolimus-induced intracellular calcium leak may attenuate endothelial dysfunction and the consequent hypertension.

Inflammation, Oxidative Stress, and Repair Capacity of the Vascular Endothelium in Obstructive Sleep Apnea

Indirect evidence implicates endothelial dysfunction in the pathogenesis of vascular diseases associated with obstructive sleep apnea (OSA). We investigated directly whether dysfunction and inflammation occur in vivo in the vascular endothelium of patients with OSA. The effects of continuous positive airway pressure (CPAP) therapy on endothelial function and repair capacity were assessed. Thirty-two patients with newly diagnosed OSA and 15 control subjects were studied. Proteins that regulate basal endothelial nitric oxide (NO) production (endothelial NO synthase [eNOS] and phosphorylated eNOS) and inflammation (cyclooxygenase-2 and inducible NOS) and markers of oxidative stress (nitrotyrosine) were quantified by immunofluorescence in freshly harvested venous endothelial cells before and after 4 weeks of CPAP therapy. Vascular reactivity was measured by flow-mediated dilation. Circulating endothelial progenitor cell levels were quantified to assess endothelial repair capacity. Baseline endothelial expression of eNOS and phosphorylated eNOS was reduced by 59% and 94%, respectively, in patients with OSA compared with control subjects. Expression of both nitrotyrosine and cyclooxygenase-2 was 5-fold greater in patients with OSA than in control subjects, whereas inducible NOS expression was 56% greater. Expression of eNOS and phosphorylated eNOS significantly increased, whereas expression of nitrotyrosine, cyclooxygenase-2, and inducible NOS significantly decreased in patients who adhered to CPAP > or = 4 hours daily. Baseline flow-mediated dilation and endothelial progenitor cell levels were lower in patients than in control subjects, and both significantly increased in patients who adhered to CPAP > or = 4 hours daily. OSA directly affects the vascular endothelium by promoting inflammation and oxidative stress while decreasing NO availability and repair capacity. Effective CPAP therapy is associated with the reversal of these alterations.

Arsenite Increases Vasoconstrictor Reactivity in Rat Blood Vessels: Role of Endothelial Nitric Oxide Function

Arsenite has been shown to inhibit endothelium-dependent, nitric oxide-mediated vasodilation in vitro. This study investigated the effects of arsenite on vascular reactivity in vivo. Saline or sodium arsenite (6 mg kg-1) was administered intravenously in Wistar-Kyoto rats for 4 h. As compared to saline, arsenite significantly increased vasoconstrictor responses to phenylephrine in both rat isolated aorta and renal arteries examined in tissue bath. This change was diminished after preincubation of the tissues with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, which increased phenylephrine-induced vasoconstriction to a similar extent as arsenite. In contrast, acetylcholine-induced vasodilation, mediated by nitric oxide in the aorta and by an endothelium-derived hyperpolarizing factor in renal arteries, was not affected by arsenite. Arsenite induced expression of heat shock proteins Hsp72, Hsp32, and Hsp90, but endothelial nitric oxide synthase expression was not changed. The effects of arsenite on vasoreactivity were unlikely to be mediated by heat shock protein induction, because blockade of heat shock protein induction had little effect on the increased vasoconstriction in vessels from arsenite-treated animals. Our study suggests that in vivo arsenic treatment increases vasoconstrictor reactivity by compromising basal endothelial nitric oxide function, which is not caused by altered endothelial nitric oxide synthase expression.

Relationship between changes in brachial artery flow-mediated dilation and basal release of nitric oxide in subjects with Type 2 diabetes

Assessment of flow-mediated dilation (FMD) after forearm ischemia is widely used as a noninvasive bioassay of stimulated nitric oxide (NO)-mediated conduit artery vasodilator function in vivo. Whether this stimulated endothelial NO function reflects basal endothelial NO function is unknown. To test this hypothesis, retrospective analysis of randomized crossover studies was undertaken in 17 subjects with Type 2 diabetes; 9 subjects undertook an exercise training or control period, whereas the remaining 8 subjects were administered an angiotensin II receptor blocker or placebo. FMD was assessed by using wall tracking of high-resolution brachial artery ultrasound images in response to reactive hyperemia. Resistance vessel basal endothelium-dependent NO function was assessed by using intrabrachial administration of NG-monomethyl-L-arginine (L-NMMA) and plethysmographic assessment of forearm blood flow (FBF). FMD was higher after intervention compared with control/placebo (6.15+/-0.53 vs. 3.81+/-0.72%, P<0.001). There were no significant changes in the FBF responses to L-NMMA. Regression analysis between FMD and L-NMMA responses at entry to the study revealed an insignificant correlation (r=-0.10, P=0.7), and improvements in FMD with the interventions were not associated with changes in the L-NMMA responses (r=-0.04, P=0.9). We conclude that conduit artery-stimulated endothelial NO function (FMD) does not reflect basal resistance vessel endothelial NO function in subjects with Type 2 diabetes.

Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells

Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that approximately 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis.

Basal Endothelial Nitric Oxide Release Is Preserved in Overweight and Obese Adults

Impaired basal nitric oxide release is associated with a number of cardiovascular disorders including hypertension, arterial spasm, and myocardial infarction. We determined whether basal endothelial nitric oxide release is reduced in otherwise healthy overweight and obese adult humans. Seventy sedentary adults were studied: 32 normal weight (BMI <25 kg/m(2)), 24 overweight (BMI > or = 25 < 30 kg/m(2)), and 14 obese (BMI > or = 30 kg/m(2)). Forearm blood flow (FBF) responses to intra-arterial infusions of N(g)-monomethyl-L-arginine (5 mg/min), a nitric oxide synthase inhibitor, were used as an index of basal nitric oxide release. N(g)-monomethyl-L-arginine elicited significant reductions in FBF in the normal weight (from 4.1 +/- 0.2 to 2.7 +/- 0.2 mL/100 mL tissue/min), overweight (4.1 +/- 0.1 to 2.8 +/- 0.2 mL/100 mL tissue/min), and obese (3.9 +/- 0.3 to 2.7 +/- 0.2 mL/100 mL tissue/min) subjects. Importantly, the magnitude of reduction in FBF (approximately 30%) was similar among the groups. These results indicate that the capacity of the endothelium to release nitric oxide under basal conditions is not compromised in overweight and obese adults.

Asymmetrical Dimethylarginine Plasma Concentrations Are Related to Basal Nitric Oxide Release but Not Endothelium-Dependent Vasodilation of Resistance Arteries in Peritoneal Dialysis Patients

Vascular dysfunction in chronic renal failure may be linked to reduced nitric oxide (NO) bioactivity and increased circulating concentrations of the endogenous NO synthase inhibitor asymmetrical dimethyl L-arginine (ADMA). The association between ADMA and basal endothelial NO release and endothelium-dependent vasodilation in resistance arteries of chronic renal failure patients is unknown. Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine, the endothelium-independent vasodilator nitroglycerine, and the endothelium-dependent vasoconstrictor N(G)-monomethyl-L-arginine (L-NMMA) were assessed in 37 peritoneal dialysis patients. L-arginine and ADMA plasma concentrations were measured by HPLC. ADMA (mean +/- SEM: 0.68 +/- 0.02 micromol/L) was associated with basal forearm blood flow (r = -0.33; P < 0.05) and L-NMMA induced vasoconstriction (r = -0.55; P < 0.0005), but not with dilator effects of acetylcholine or nitroglycerine. L-arginine (68 +/- 3 micromol/L) tended to correlate with acetylcholine-induced vasodilation (r = 0.32; P = 0.05) but was not associated with other parameters. ADMA is related to basal but not to acetylcholine-stimulated NO bioactivity in patients on peritoneal dialysis. Impaired endothelium-dependent vasodilation found in chronic renal failure is not explained by elevated circulating NO synthase inhibitors in renal failure.

Medroxyprogesterone acetate does not antagonize estrogen-induced increases in endothelium-dependent vasodilation: potential clinical implications

Objective: Determine the effect of combined 17β-estradiol benzoate (E 2) and medroxyprogesterone acetate (MPA) administration on endothelium-dependent vasorelaxation to acetylcholine (ACh). Design: Prospective, ex vivo study. Setting: Academic research laboratory. Animal(s): Mature female rats. Intervention(s): Ovariectomized rats received one of the following interventions daily for 3 days: [1] corn oil via IM injection, [2] E 2 (20 μg/kg IM), or [3] E 2 (20 μg/kg IM) and MPA (10 mg/kg IM). Main Outcome Measure(s): Basal release of nitric oxide (NO) and endothelium-dependent and endothelium-independent vasodilation from thoracic aortas obtained from each group. Result(s): Estradiol treatment potentiated the endothelium-dependent relaxation to ACh when compared with the control group. Administration of MPA with E 2 did not antagonize the beneficial effect of E 2 on endothelium-dependent relaxation. Estradiol treatment alone or in combination with MPA did not affect endothelium-independent vasodilation as compared with the case of the control group. Administration of E 2 resulted in increased basal NO release (assessed indirectly by measuring the constrictor response to N G-nitro- l-arginine [methyl ester (L-NAME)]) when compared with the case of the control group, and the addition of MPA to E 2 did not alter the effect of E 2 on basal NO release. Conclusion(s): Estradiol potentiates endothelium-dependent relaxant responses and increases basal endothelial NO release. Medroxyprogesterone acetate does not antagonize these effects of E 2.