Basal Nitric Oxide Production Research Articles

Intermittent hypoxia conditioning improves basal and stimulated nitric oxide production in spontaneously hypertensive rats

Earlier we reported that intermittent hypoxia conditioning (IHC) slows development of hypertension in young spontaneously hypertensive rats (SHR) and stimulates nitric oxide (NO) synthesis. Now we have compared basal and stimulated NO production in IHC and sham conditioned, 4–5 week old SHR. IHC protocol: 20 days, 5 to 8 cycles of 9.5–10% O2, each 5 to 10 min, separated by 4 min normoxia. Sham protocol: same regimen but no change in inspired O2. The 20 day rise of systolic BP of IHC rats was less than in sham rats (from 131±2 mmHg to 162±2 mmHg vs 202±3 mmHg in sham rats, P<0.001). Basal NO production was evaluated by the contractile response to norepinephrine (NE, 10− 8M) of isolated aortic rings in the presence and absence of L‐NAME (10−3 M), an NO synthase inhibitor. After L‐NAME, NE‐induced contraction increased more (P<0.05) in IHC rats (66±16%) than in sham rats (35±3%). Endothelium‐dependent relaxation (EDR) was evaluated by the response to acetylcholine of NE‐precontracted rings. In 8‐week old IHC rats, EDR was not impaired and did not differ from that of 4–5 week old SHR (60±6%). EDR was impaired in 8‐week old sham‐IHC rats compared to 4–5 week old rats (28±6 vs 55±5%, P<0.002). Therefore, the antihypertensive effect of IHC is associated with improvement of both basal and stimulated NO production. These effects may indicate an important role of NO in the antihypertensive action of IHC. (Support: UNTHSC Cardiovasc. Res. Inst.)

Gender differences in aortic endothelial function of streptozotocin‐induced diabetic rats: possible involvement of protein kinase C beta and nitric oxide production

Little is known of the interaction between diabetes and gender in the vasculature. The objective of this study was to investigate whether there are gender differences in rat aortic endothelial function in streptozotocin (STZ, 60mg/kg, iv)‐induced diabetes and the roles of protein kinase C beta (PKCβ) and nitric oxide (NO). Endothelium‐dependent vasodilatation (EDV) to acetylcholine (ACh; 10−8 to 10−5M) was measured in aortic rings precontracted with phenylephrine (PE; 2μM) before and after pretreatment with LY341684 (1μM), a selective PKCβ inhibitor. Constrictor response curves to PE (10−8 to 10−5M) were also generated before and after incubation with L‐NAME (200μM), an endothelial nitric oxide synthase inhibitor. STZ‐induced diabetes impaired aortic EDV to ACh only in females. Inhibition of PKCβ increased the sensitivity to ACh in both control and diabetic male rats. However, PKCβ inhibition enhanced the sensitivity to ACh only in aorta taken from diabetic female rats. Addition of L‐NAME resulted in a significant potentiation of the contractile responses to PE in all groups. However, aorta from control females had a greater maximal potentiation of the PE responses than others. These data suggest that the predisposition of female rat aorta to vascular injury in diabetes is possibly due to differences in enhanced PKCβ activation and decreased basal NO production (supported by NIH/NIDCR).

Activation of Nrf‐2 Enhances the Function of Human Renal Glomerular Endothelial Cells

Diabetes causes nitric oxide (NO) deficiency dysfunction and nephropathy. Recently, bardoxolone methyl, an activator of nuclear E2‐related factor 2 (Nrf2)–antioxidant response element (ARE) pathway was shown to increase the GFR in diabetic nephropathy, but the mechanism was not established. We detected 1 to 3 potential AREs in the human dimethylarginine dimethylaminohydrolase (DDAH‐1 and ‐2) gene promoter regions. Since DDAH metabolizes the endogenous NOS inhibitor ADMA and upregulates eNOS, we tested the hypothesis that activation of Nrf‐2 enhances endothelial function. Incubation of human renal glomerular endothelial cells (HRGECs) with the Nrf2 activator, tert‐butylhydroquinone (tBHQ; 5–20uM) dose‐dependently and significantly (P<0.05) increased medium nitrite(69±3.7%), NO (62±17.1%) and DDAH activity (47±8.5%), and increased endothelial eNOS, DDAH‐1 and‐2 protein expression by 43±17.1%, 62±20.9% and 48±17.6% (P<0.05). tBHQ markedly increased Nrf‐2 protein in the cell nuclear fraction. In contrast, siRNA knockdown of Nrf‐2, compared tocontrol siRNA, abolished all these tBHQ‐mediated effects and attenuated basal cell proliferation, nitrite and NO production, DDAH activity, and eNOS and DDAH protein expression. In conclusion, activation of Nrf‐2 enhanced endothelial function by simultaneously increasing expression of eNOS and DDAH, thereby increasing NO generation and activity that leads to endothelial cell proliferation. These actions might explain the beneficial effects of Nrf‐2 activation in human diabetic nephropathy.

Nitric Oxide Supplementation in Postischemic Acute Renal Failure: Normotension Versus Hypertension

Nitric oxide (NO) has been suggested to play a pivotal role in ischemic acute renal failure (ARF) but there are controversies about its role in hypertensive and non hypertensive ischemic kidney. Multiple strategies including administration of exogenous NO donors have been shown to protect the kidney against toxic or ischemic injury, suggesting endothelial dysfunction as impaired NO generation due to ischemia. However, in postischemic kidney, NO derived from inducible nitric oxide synthase (iNOS) has been considered to enhance the tissue damage while iNOS inhibition decreased the tubular damage. It is well known decrease in basal production of NO in essential hypertension and that long lasting hypertension damages medium size and small-size blood vessels, therefore predisposes nephroangiosclerosis patients to ARF. Many studies have shown that long term stimulation of NO release in normotension improves renal haemodymnamics and kidney function in ischemic form of ARF. On the other hand, there are studies that have shown that NO synthesis stimulation has no effect or even worsens tubular damage in postischemic hypertensive kidney. Therefore, it seems likely that NO supplementation plays different role in postischemic renal damage development, beneficial in well preserved normotensive kidney and limited in postischemic hypertensive kidney due to disturbed tubuloglomerular response, vasoreactivity and kidney vascular structure.

Nitric oxide has a role in attenuating the neuroendocrine response to anaphylactoid stress during lactation

Stress increases nitric oxide (NO) production in the paraventricular nucleus of the hypothalamus (PVH). Lactation diminishes the response to stress and increases basal NO production markers in the PVH of the dam. This study investigated whether lactation modified the anaphylactic reaction to egg white (EW) injection, and if nitric oxide regulates the neuroendocrine response to this stressor. The activational response of PVH to EW was assessed by c-Fos immunohistochemistry, and NO production was determined by histological staining of NADPH-diaphorase and neuronal nitric oxide synthase (nNOS) and by measuring the concentration of total nitrates and nitrites (NOx) in the hypothalamus of lactating and diestrus rats. EW injection significantly increased the number of Fos-positive neurons in the parvocellular subdivision of the PVH in diestrus, but not in lactating rats. Similarly, EW injection increased the number of NADPH-diaphorase- and nNOS-positive cells in the PVH of diestrus rats, but it did not alter the already increased basal number of NO-positive cells in lactating rats. Furthermore, the total concentration of NOx in the hypothalamus, the circulating level of corticosterone and interleukin-6 increased significantly after EW in diestrus, but not in lactating rats, compared to their corresponding controls. Intracerebral administration of L-NAME, a general NOS inhibitor, reversed the attenuation of the activational response to EW in the PVH of lactating rats. The present results show that lactation diminishes the anaphylactoid reaction to EW compared to that in diestrus rats. This attenuation was absent after L-NAME treatment, suggesting that sustained NO production in the PVH during lactation may limit the neuroendocrine response to stress.

A Endothelial cell nitric oxide bioavailability and insulin sensitivity are regulated by IGF-1 and insulin receptor levels

BackgroundIn a similar manner to insulin, the growth promoting hormone Insulin-like Growth Factor-1 (IGF-1), may be an important regulator of endothelial nitric oxide (NO) bioavailability. We have previously reported evidence...

The relationship between sports engagement, body mass index and physical abilities in children

Hypokinesia, which is a consequence of modern technological development, is now one of the three biggest health risk factors. Whereas physical inactivity and obesity in adults is subject of concern, these factors in children presage an even greater public health problem because many health-related problems and life-threatening diseases begin in childhood and adolescence. It has been noted that regular physical activity modulates vascular endothelial function via an effect on basal nitric oxide (NO) production, thus leading to a significant reduction in the incidence of cardiovascular disease (1). Exercise can also increase energy expenditure and can create a negative energy balance. Conversely, scientific data regarding the effect of school-based physical activity interventions on body mass index (BMI) in children are conflicting (2, 3). Moreover, there is insufficient clinically useful scientific data on BMI in children and its possible association with motor skills. With both sexes, motor abilities improve from physical development in adolescents, and regular sports training influences fitness and motor abilities. The aim of our study was to evaluate the morphological status and physical abilities of 13-year-old children in order to investigate the relationship between sports engagement, BMI and physical abilities. The results of our study should give coaches important parameters in the selection of young athletes, as well as provide them with information that may be useful in training and programming assignments.

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.

Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose

Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations. Endothelium-dependent relaxation was studied in aortic rings from Sprague-Dawley rats. We also used cultured rat aortic and human umbilical vein endothelial cells. The MG was measured by HPLC and Western blotting and assay kits were used. Incubation of aortic rings with MG (30 µM) or high glucose (25 mM) attenuated endothelium-dependent, acetylcholine-induced relaxation, which was restored by two different MG scavengers, aminoguanidine (100 µM) and N-acetyl cysteine (NAC) (600 µM). Treatment of cultured endothelial cells with MG or high glucose increased cellular MG levels, effects prevented by aminoguanidine and NAC. In cultured endothelial cells, MG and high glucose reduced basal and bradykinin-stimulated nitric oxide (NO) production, cGMP levels, and serine-1177 phosphorylation and activity of endothelial NO synthase (eNOS), without affecting threonine-495 and Akt phosphorylation or total eNOS protein. These effects of MG and high glucose were attenuated by aminoguanidine or NAC. Our results show for the first time that MG reduced serine-1177 phosphorylation, activity of eNOS and NO production. MG caused endothelial dysfunction similar to that induced by high glucose. Specific and safe MG scavengers have potential to prevent endothelial dysfunction induced by MG and high glucose concentrations.

A Fluorescence‐Activated Cell Sorter Analysis of the Relationship Between Protein Kinase G and Endothelial Nitric Oxide Synthase

The process of regulation of NOS after production of nitric oxide is not yet delineated. Protein kinase G may exert a feedback regulation of this enzyme. We used diaminofluorescein assays to detect changes in basal nitric oxide production caused by modulators of protein kinase G activity in freshly isolated ovine lung microvascular endothelial cells. We also used fluorescence activated cell sorter analysis (FACS) to determine molecular and phosphorylation changes caused by PKG activation with 8-Br-cGMP. The PKG activator, 8-Br-cGMP (100 μM) produced a shift in the basal NO production curve downward. The inhibition began within 5 min and was sustained over 4.5 hr. The two protein kinase G inhibitors 100 μM Rp-8-Br-PET-cGMPS and 50 nM guanosine 3'-5'-cyclic monophosphoro thionate-8-Br-Rp isomer Na salt and the cGMP inhibitor 4 μM Rp-8-pCPT-cGMPS all enhanced NO production as seen by the upward shift in the basal NO curve. Conversely, the PKG activator drug, 100 μM guanosine-3'-5'-cyclic monophosphate-β-phenyl-1NF-ethano-8-bromo sodium salt decreased NO production causing a downward shift in the basal curve. FACS analysis revealed that 5 μM 8-Br-cGMP in <5 min caused an increase in N-terminal labeling of NOS and a decrease in both C-terminal and serine 1177 labeling of NOS. 8-Br-cGMP appeared to increase PKG 1α and to decrease PKG 1β labeling. Changes in other phosphorylation sites were less consistent but overall mean channel fluorescence increased from 19.92 to 217.36 for serine 116 and decreased from 329.27 to 254.03 for threonine 495 phosphorylation. Data indicated that PKG caused both molecular and phosphorylation changes in NOS.

Leaf senescence signaling: the Ca2+-conducting Arabidopsis cyclic nucleotide gated channel2 acts through nitric oxide to repress senescence programming.

Ca(2+) and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca(2+). The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca(2+) modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca(2+) and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca(2+) uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca(2+) accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H(2)O(2) generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca(2+) channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca(2+) signaling provides evidence consistent with genetic studies of the relationship between Ca(2+) signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca(2+) uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.

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.

Selective in vivo antagonism of endothelin receptors in transforming growth factor-β1 transgenic mice that mimic the vascular pathology of Alzheimer’s diseaseThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Increased levels of transforming growth factor-beta1 (TGF-beta1) induce a vascular pathology that shares similarities with that seen in Alzheimer's disease, and which possibly contributes to the cognitive decline. In aged transgenic mice that overexpress TGF-beta1 (TGF mice), we previously found reduced dilatory function and selectively impaired endothelin-1 (ET-1)-induced contraction. Here we studied the effects of chronic treatments with selective ETA (ABT-627) or ETB (A-192621) receptor antagonist on cerebrovascular reactivity, cerebral perfusion, or memory performance. The dilatory deficit of TGF mice was not improved by either treatment, but both ET-1 contraction and basal nitric oxide (NO) production were distinctly altered. Although ABT-627 was devoid of any effect in TGF mice, it virtually abolished the ET-1-induced contraction and NO release in wild-type (WT) littermates. In contrast, A-192621 only acted upon TGF mice with full recovery of ET-1 contraction and baseline NO synthesis. TGF mice, treated or not, had no cognitive deficit in the Morris water maze, nor did ABT-627-treated WT controls despite severely impaired vasoreactivity. These findings confirm that ETA receptors primarily mediate the ET-1-induced contraction. Further, they suggest that ETB receptors play a detrimental role in conditions of increased TGF-beta1 and that vascular dysfunction does not inevitably lead to cognitive deficit.

Quantification of the In Vivo Production of Nitric Oxide in the Rat Brainstem

Pharmacological evidence suggests that nitric oxide (NO) functions as a neuromodulator within the nucleus tractus solitarius (NTS) of the medulla oblongata contributing to the regulation of sympathetic efferent output at rest and during exercise. It is well known that NO is formed through the oxidation of L-arginine by nitric oxide synthase (NOS) within the brainstem. However, due to the instability and short half-life of NO, there are currently no methods to directly measure the in vivo production of the molecule. As a result, it is difficult to draw definitive conclusions with regard to the role NO plays in modulating sympathetic outflow. PURPOSE: To develop a technique that allows quantification of the in vivo production of NO within the NTS using the fluorescent probe 1,2-diaminoanthraquinone (DAA) previously utilized primarily in vitro. DAA reacts with NO to form a water-soluble red-fluorescent precipitate. Thus fluorescence intensity is indicative of NO production. METHODS: In male decerebrate Sprague-Dawley rats (n=24), DAA (100 μg/mL) was microdialyzed into the NTS for 5, 10, 15, and 30 minutes with and without the nonspecific NOS inhibitor L-NAME (1 mM) or the NO precursor L-arginine (1 μM or 10 μM). Following these procedures, animals underwent fixation via transcardial perfusion with paraformaldehyde. Subsequently, medullary tissue was harvested and prepared for microscopic viewing. Fluorescence intensity was quantified using ImageJ software. RESULTS: Basal NO production increased progressively after 5, 10, 15, and 30 minutes of DAA microdialysis. As expected, the dialysis of L-arginine within the NTS produced time and dose dependent elevations in NO production in vivo as quantified by post-mortem analysis of brainstem tissue. In contrast, the dialysis of L-NAME significantly reduced NO production within the NTS at all time points as reflected by decreased fluorescence in fixed tissue. CONCLUSION: These studies demonstrate that the innovative use of an established in vitro technique can be adopted to quantify the in vivo production of NO. In the future, use of this developed application could serve as an important tool for determining the role of NO within the NTS in regulating sympathetic nerve activity at rest and during exercise. Supported by HL-094075 and HL-088422.

Effect of high soy diet on the cerebrovasculature and endothelial nitric oxide synthase in the ovariectomized rat

High soy (HS) diets are neuroprotective and promote vascular dilatation in the periphery. We hypothesized that an HS diet would promote vascular dilatation in the cerebrovasculature by mimicking estradiol's actions on the endothelial nitric oxide synthase (eNOS) system including increasing eNOS expression and decreasing caveolin-1 expression to increase nitric oxide (NO) production. Ovariectomized rats were fed HS or a soy-free diet (SF) ± low physiological estradiol (E2) for 4 weeks. Neither E2 nor HS altered middle cerebral artery (MCA) structure or vascular responses to acetylcholine, serotonin, or phenylephrine. Estradiol enhanced bradykinin-induced relaxation in an eNOS-dependent manner. Although E2 and HS increased eNOS mRNA expression in the brain and cerebrovasculature, they had no effect on eNOS protein expression or phosphorylation in the MCA. However, E2 decreased caveolin-1 protein in the MCA. In MCAs neither E2 nor HS altered estrogen receptor (ER) alpha expression, but E2 did reduce ER beta levels. These data suggest that HS diets have no effect on vascular NO production, and that E2 may modulate basal NO production by reducing the expression of caveolin-1, an allosteric inhibitor of NOS activity. However, the effects of E2 and HS on the cerebrovasculature are small and may not underlie their protective actions in pathological states.

Oestrogenic Compounds Modulate Cytokine-induced Nitric Oxide Production in Mouse Osteoblast-like Cells

Nitric oxide (NO) is a mediator of bone metabolism with effects on both bone resorption and formation. Its production by both the constitutive and inducible isoforms of nitric oxide synthase (NOS) is affected by oestrogen in several types of cell and in tissues other than bone cells. Recently, oestrogens were found to increase basal NO production by osteoblasts via enhanced activity or expression, or both, of NOS-3. Inflammatory cytokines, however, increase NO by increasing the expression of NOS-2. In this study we have examined whether cytokine-induced NO production by osteoblastic cells was affected by oestrogenic compounds by studying the effect of 17beta-oestradiol and the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen on cytokine-induced NO production in oestrogen receptor positive MC3T3-E1 osteoblast-like cells. Combinations of the inflammatory cytokines interleukin-1beta, tumour necrosis factor-alpha, and interferon-gamma with lipopolysaccharide stimulated NO production up to 11-fold. This cytokine-induced NO production was further increased dose-dependently by the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen (133.3 +/- 3.2% and 146.0 +/- 13.2%, respectively). 17Beta-oestradiol either had no effect on or slightly inhibited cytokine-induced NO production. It did, however, dose-dependently counteract the stimulatory effect of the anti-oestrogens. Concentrations of 17beta-oestradiol needed to prevent the stimulatory effect of 4-hydroxytamoxifen were ca tenfold that of ICI164,384. These findings show that, in addition to the stimulatory effect of oestrogen on basal NO production by NOS-3, cytokine-induced NO production is also affected by oestrogenic compounds in osteoblasts.

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.

Cyclin-Dependent Kinase 5 Phosphorylates Endothelial Nitric Oxide Synthase at Serine 116

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Change in Augmentation Index during NOS Inhibition, an Index of Basal NO Production, Is an Independent Determinant of Large-Artery Function

Background/Aims: Arterial wave reflection, measured as augmentation index (AIx), and central pulse pressure (PP) closely predict cardiovascular events. We hypothesized that basal nitric oxide (NO) production would be a determinant of AIx and central PP. Methods: AIx and central PP were assessed at baseline by pulse wave analysis in 86 male subjects across a wide range of age, blood pressure and lipid values. The basal NO production in the cardiovascular system was then determined as change in AIx during NO synthase blockade with N^G-monomethyl-L-arginine (L-NMMA, 3.25 mg/kg). Results: AIx increased from 17.5 ± 14.6 to 23.1 ± 14.2 during L-NMMA infusion (p < 0.001). The increase in AIx during NO synthase blockade, an index of basal NO production, was inversely related to baseline central AIx and PP, and positively to PP amplification. Multiple linear regression analyses disclosed that in addition to age and mean blood pressure, change of AIx to L-NMMA is a strong and independent determinant of baseline central AIx, central PP and PP amplification. Conclusion: Greater change of AIx to L-NMMA, an index of basal NO production, is associated with better large-artery function. Therefore, therapeutic interventions which increase the basal NO production might be particularly effective in reducing cardiovascular risk.

Angiostatin Inhibition of Vascular Endothelial Growth Factor– Stimulated Nitric Oxide Production in Endothelial Cells

Angiostatin (AS), a proteolytic fragment of plasminogen, is a potent antiangiogenic factor. It was reported that AS attenuates the vasodilatory response to vascular endothelial growth factor (VEGF) in isolated interventricular arterioles. Here, we investigated the effect of AS on nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). AS inhibited VEGF-stimulated NO production in a dose-dependent manner, whereas AS alone did not affect basal NO production. Disruption of kringle structures by reduction of disulfide bonds resulted in the loss of the inhibitory effect of AS on VEGF-stimulated NO production. To elucidate how AS might impair VEGF activation of endothelial NO synthase (eNOS), we further examined whether AS would affect Ca2+-dependent and -independent pathways of eNOS activation. AS had no effect on the transient increase in cytosolic Ca2+ levels elicited by VEGF. In contrast, AS prevented VEGF-potentiated eNOS phosphorylation at Ser1177. These results clearly indicate that AS inhibits VEGF-stimulated NO production in HUVECs without affecting basal NO production. The kringle structures of AS are required for this effect, and impairment of Ser1177 phosphorylation of eNOS might be involved in the inhibition of VEGF-stimulated NO production by AS.