Vascular NO Production Research Articles

Regulation of endothelial NO by DDAH1 and DDAH2 isoforms

Increased levels of the endogenous eNOS inhibitor, ADMA, has been implicated in the endothelial dysfunction associated with cardiovascular disease. Endothelial ADMA and L‐NMMA are metabolized by the enzyme Dimethylarginine Dimethylamino hydrolase (DDAH). DDAH has two isoforms, both of which are expressed in the endothelium. However, the role of these isoforms in regulating vascular NO production is unclear. Therefore, studies were performed to study the effects of DDAH gene silencing and over‐expression on eNOS derived NO production using BAECs. DDAH‐1 over‐expression increased NO production by 21%, while DDAH‐2 over‐expression had no effect. DDAH‐1 gene silencing resulted in a marginal decrease in NO production while DDAH‐2 gene silencing resulted in a 58% decrease in NO production. The DDAH‐2 gene silencing effect on NO was partially restored with L‐arginine supplementation to 65% of the control. HPLC measurements of intracellular ADMA revealed no significant increase in ADMA levels following DDAH gene silencing. However, western blot analysis revealed a 40% reduction in eNOS expression following DDAH‐2 gene silencing. These results demonstrate that DDAH‐2 can modulate endothelial NO bioavailability through ADMA independent mechanisms involving decreased eNOS expression. These finding demonstrate a novel mechanism through which DDAH regulates endothelial NO generation.

Impaired nerve-mediated relaxation of penile tissue from caveolin-1 deficient mice

Caveolin-1-deficient mice are characterised by a high vascular NO production. Because NO-dependent smooth muscle relaxation is considered to play an important role in penile erection, it was hypothesized that the erectile function would be affected by genetic ablation of caveolae. This study assessed penile erectile mechanisms in caveolin-1 knockout (KO) mice ex vivo. Immunofluorescence confirmed caveolin-1 expression primarily in the endothelium surrounding the sinusoids of the corpus cavernosum, but also in smooth muscle cells of the sinusoidal bundles. In KO mice, caveolin-1 was absent, and the expression of the caveola-associated protein PTRF-Cavin was reduced. Nitric oxide synthase (endothelial and neuronal) and caveolin-3 levels were not affected, and staining of the neuronal marker PGP 9.5 did not disclose any apparent change in the density or pattern of innervation. Moreover, no apparent morphological differences were noted. Functionally, the force response following stimulation of α 1-adrenergic receptors, and the sensitivity to the Rho-kinase inhibitor Y27632, were unaltered, whereas relaxation of α 1-precontracted corpus cavernosum in response to electrical field stimulation and the muscarinic agonist carbachol were impaired. The nitric oxide donor sodium nitroprusside produced less relaxation in KO as compared to wild type corpus cavernosum. We conclude that nerve-mediated dilatation of the corpus cavernosum is impaired in the absence of caveolin-1, and that this is due in part to reduced sensitivity of the target tissue to NO. All in all our data support an important role of caveolin-1 in penile erection.

Endothelial Arginase II

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E-null (ApoE(-/-)) and wild-type mice fed a high cholesterol diet. In ApoE(-/-) mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II(-/-) mice) prevents high-cholesterol diet-dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein-dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.

ORIGINAL ARTICLE: Lack of Consistent Association Between Endothelial Nitric Oxide Synthase Gene Polymorphisms, Homocysteine Levels and Recurrent Pregnancy Loss in Tunisian Women

Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been associated with reduced vascular NO production or increased level of homocysteine, and evaluated as risk factors for recurrent pregnancy loss (RPL). Therefore, in this case-control study, we aimed to determine the effects of some eNOS functional polymorphisms: the 27-bp intron 4 repeat, the 894G/T of exon 7, and the promoter substitution -786T/C, in women with RPL. We genotyped 350 patients with RPL and 200 healthy women by polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). The homocysteine total plasma concentrations (tHcy) were determined by enzyme-linked immunosorbent assay (ELISA). None of the eNOS polymorphisms-related alleles, genotypes, and haplotypes were associated with RPL. The tHcy were similar between patients and controls; no significant association between tHcy levels and eNOS genotypes could be evidenced. The present study identified a lack of association between eNOS gene polymorphisms, the risk of RPL and tHcy levels.

Abstract 1134: Crucial Role of Cu/Zn-SOD in the Synthesis of Endothelium-Derived Hyperpolarizing Factor (EDHF) during Reactive Hyperemia in Mouse Mesenteric Microcirculation in Vivo

Background: We have recently demonstrated that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role as an EDHF synthase. In this study, we examined the possible role of Cu/Zn-SOD in the synthesis of EDHF/H 2 O 2 during reactive hyperemia (RH) in mouse mesenteric microcirculation in vivo. Methods: Mesenteric arterioles from wild-type (wild) and Cu/Zn-SOD −/− mice (SOD-KO) (n=5 each, 22– 60 μm) were continuously observed with a pencil-typed intravital microscope (×600 magnification) during acetylcholine (ACh)-induced vasodilatation and RH (reperfusion after 60 sec of mesenteric artery occlusion) under cyclooxygenase blockade (indomethacin) under the following 3 conditions; control, NO synthase inhibitor (L-NMMA) and L-NMMA+catalase (a specific decomposer of H 2 O 2 ). Results: In mesenteric arterioles of wild-type mice, endothelium-dependent relaxations to ACh (34±4%) and vasodilatation during RH (40±5%) were resistant to L-NMMA (ACh 23±7% and RH 20±5%, both P<0.05), but were markedly inhibited by L-NMMA+catalase (ACh, 8±2% and RH, 4±3%, both P<0.01 vs. control). In mesenteric arterioles of SOD-KO mice, L-NMMA markedly inhibited the relaxations to ACh (30±4% to 8±2%, P<0.01) and vasodilatation during RH (40±5% to 5±2%, P<0.01), and the responses were not inhibited by the addition of catalase (ACh, 3±2% and RH, 4±2%, not significant vs. control). In the presence of L-NMMA, RH-induced increase in blood flow was 330±5% of baseline in the wild-type mice, whereas in the SOD-KO mice, the response was significantly reduced to 145±23% (P<0.01). In the mesenteric arterioles of SOD-KO mice, tempol, an SOD mimetic, significantly increased the ACh-induced vasodilation, which effect was abolished by catalase. Vascular H 2 O 2 production in response to ACh in mesenteric arterioles (fluorescent method) was significantly increased in wild-type mice but was markedly impaired in SOD-KO mice. By contrast, vascular NO production was significantly increased in wild-type mice and was unaltered in SOD-KO mice. Vascular level of BH 4 in the aorta was comparable between the two genotypes. Conclusions: These results indicate that endothelial Cu/Zn-SOD plays an important role as an EDHF synthase during RH in mouse mesenteric microcirculation in vivo.

Abstract 1420: Mitochondrial Arginase II Constrains Endothelial NOS Activity

Background: Arginase II is known as a major isoenzyme in the vascular endothelial cells of humans and other species and modulates endothelial nitric oxide synthase (eNOS) activity by regulating intracellular L-arginine bioavailability. We tested under hypothesis that subcellular localization of arginase II is confined to the mitochondria and mitochondrial arginase II deficiency reciprocally regulates the vascular endothelial NO production, contributes to improve endothelial dysfunction and decrease vascular stiffness. Methods and Results: Western blot, immunocytochem with Mitotracker and immunoEM confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly reduced, whereas NO production was significantly increased in aorta and isolated endothelial cells from arginase II knock out (ArgII−/−) compared to WT mice. Vasorelaxation to acetylcholine (ACh) was markedly enhanced and vasoconstriction to phenylephrine (PE) was impaired in ArgII−/− rings. Furthermore inhibition of eNOS by N G -nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored PE response. The baseline pulse wave velocity was significantly decreased in ArgII −/− mice (3.30 ± 0.21 vs. 3.64 ± 0.12 m/s, ArgII −/− vs. WT, *p<0.01), while 14 days of L-NAME treatment resulted in significant increase the PWV in both WT (4.23 ± 0.11 m/s, **p<0.001) and ArgII −/− mice (4.36 ± 0.20 m/s, **p<0.001). Conclusions: These data suggest that arginase II is predominantly confined to the mitochondria and this mitochondrial arginase II regulate the NO production, vascular endothelial function, and vascular stiffness by modulating eNOS activity.

Angiotensin II Stimulates Endothelial NO Synthase Phosphorylation in Thoracic Aorta of Mice With Abdominal Aortic Banding Via Type 2 Receptor

Abdominal aortic banding in mice induces upregulation of angiotensin II (Ang II) type 2 (AT2) receptors in the pressure-overloaded thoracic aorta. To clarify mechanisms underlying the vascular AT2 receptor-dependent NO production, we measured aortic levels of endothelial NO synthase (eNOS), eNOS phosphorylated at Ser633 and Ser1177, protein kinase B (Akt), and Akt phosphorylated at Ser473 in thoracic aortas of mice after banding. Total eNOS, both forms of phosphorylated eNOS, Akt, and phosphorylated Akt levels, as well as cGMP contents, were significantly increased 4 days after banding. The administration of PD123319 (an AT2 receptor antagonist) or icatibant (a bradykinin B2 receptor antagonist) abolished the banding-induced upregulation of both forms of phosphorylated eNOS, as well as elevation of cGMP, but did not affect the upregulation of eNOS, Akt, and phosphorylated Akt. In the in vitro experiments using aortic rings prepared from banded mice, Ang II produced significant increases in both forms of phosphorylated eNOS, as well as cGMP, and these effects were blocked by PD123319 and icatibant. Ang II-induced eNOS phosphorylation and cGMP elevation in aortic rings were inhibited by protein kinase A (PKA) inhibitors H89 and KT5720 but not by phosphatidylinositol 3-kinase inhibitors wortmannin and LY24002. The contractile response to Ang II was attenuated in aortic rings from banded mice via AT2 receptor, and this attenuation was blocked by PKA inhibitors. These results suggest that the activation of AT2 receptor by Ang II induces phosphorylation of eNOS at Ser633 and Ser1177 via a PKA-mediated signaling pathway, resulting in sustained activation of eNOS.

Tetrahydrobiopterin, but Not l -Arginine, Decreases NO Synthase Uncoupling in Cells Expressing High Levels of Endothelial NO Synthase

Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. Nomega-nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4.

Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome

Cholangiocyte Endothelin 1 and Transforming Growth Factor β1 Production in Rat Experimental Hepatopulmonary Syndrome

Angiotensin Type 2 Receptor–Mediated Phosphorylation of eNOS in the Aortas of Mice With 2-Kidney, 1-Clip Hypertension

To evaluate the role of vascular angiotensin II (Ang II) type 2 (AT2) receptor in renovascular hypertension, we investigated expressions of AT2 receptor and endothelial nitric oxide synthase (eNOS) in thoracic aortas of mice with 2-kidney, 1-clip (2K1C) hypertension. The mRNA levels of AT2 receptor in aortas, but not those of AT1 and bradykinin B2 receptors, increased 14 days but not 42 days after clipping. The contractile response to Ang II (>0.1 micromol/L) was attenuated in aortic rings excised 14 days after clipping and was restored to that of rings from sham mice by antagonists of AT2 receptor (PD123319) and B2 receptor (icatibant). The aortic levels of total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), total Akt, and phosphorylated Akt at Ser473 (p-Akt) were increased in 2K1C mice on day 14, whereas only eNOS levels were increased on day 42. The aortic cGMP levels were 20-fold greater in 2K1C mice on day 14 compared with sham mice. Administration of nicardipine for 4 days before the excision of aortas 14 days after clipping not only reduced blood pressure but also decreased the aortic levels of eNOS, p-eNOS, Akt, p-Akt, and cGMP to sham levels, whereas the administration of PD123319 or icatibant to 2K1C mice decreased p-eNOS and cGMP to sham levels without affecting blood pressure and the levels of eNOS, Akt and p-Akt. These results suggest that vascular NO production is enhanced by increased eNOS phosphorylation via the activation of AT2 receptors in the course of 2K1C hypertension.

Inducible Nitric Oxide Synthase Has Divergent Effects on Vascular and Metabolic Function in Obesity

Previous studies have suggested an involvement of inducible nitric oxide synthase (iNOS) in obesity, but the relation, if any, between this and mechanisms underlying endothelial dysfunction in obesity is unknown. We studied mice fed an obesogenic high-fat or standard diet for up to 8 weeks. Obesity was associated with elevated blood pressure; resistance to the glucoregulatory actions of insulin; resistance to the vascular actions of insulin, assessed as the reduction in phenylephrine constrictor response of aortic rings after insulin preincubation (lean -21.7 +/- 11.5 vs. obese 18.2 +/- 15.5%; P < 0.05); and evidence of reactive oxygen species (ROS)-dependent vasodilatation in response to acetylcholine in aortic rings (change in maximal relaxation to acetylcholine after exposure to catalase: lean -2.1 +/- 6.0 vs. obese -15.0 +/- 3.8%; P = 0.04). Obese mice had increased expression of iNOS in aorta, with evidence of increased vascular NO production, assessed as the increase in maximal constriction to phenylephrine after iNOS inhibition with 1400W (lean -3.5 +/- 9.1 vs. obese 42.1 +/- 11.2%; P < 0.001). To further address the role of iNOS in obesity-induced vascular and metabolic dysfunction, we studied the effect of a high-fat diet in iNOS knockout mice (iNOS KO). Obese iNOS KO mice were protected against the development of resistance to insulin's glucoregulatory and vascular effects (insulin-dependent reduction in maximal phenylephrine response: obese wild-type 11.2 +/- 15.0 vs. obese iNOS KO -20.0 +/- 7.7%; P = 0.02). However, obese iNOS KO mice remained hypertensive (124.0 +/- 0.7 vs. 114.9 +/- 0.5 mmHg; P < 0.01) and had evidence of increased vascular ROS production. Although these data support iNOS as a target to protect against the adverse effects of obesity on glucoregulation and vascular insulin resistance, iNOS inhibition does not prevent the development of raised blood pressure or oxidative stress.

Loss of endothelium-dependent relaxation in abdominal aorta preserved in a co-storage system

The potentially detrimental influence of parenchymal cells on endothelial function during preservation in UW solution was examined by co-storage of rat abdominal aortic rings with isolated liver cells. Cold storage of rings in UW solution alone for up to 96 h had no effect on the response to acetylcholine, though constriction was progressively lost. Co-storage of rings with liver cells resulted in no loss of sodium nitroprusside response, but the relaxation response to acetylcholine was reduced. The loss of acetylcholine response could not be attributed to Kupffer cells, the lowering of pH, oxygen depletion, or the loss of constriction. A similar loss of endothelial function was observed in rings stored in pieces of liver, kidney or heart. We conclude that parenchymal cells exude factors during preservation by cold storage which reversibly inhibit vascular NO production. These factors could significantly impair whole organ function on reperfusion.

Asymmetric dimethylarginine produces vascular lesions in endothelial nitric oxide synthase-deficient mice: involvement of renin-angiotensin system and oxidative stress.

Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of l-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT(1) receptor antagonist), which simultaneously suppressed vascular lesion formation. These results provide the first direct evidence that the long-term vascular effects of ADMA are not solely mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of ACE and increased oxidative stress through AT(1) receptor appear to be involved in the long-term vascular effects of ADMA in vivo. This study demonstrates that asymmetrical dimethylarginine (ADMA) causes arteriosclerotic coronary lesions in mice in vivo through mechanisms other than simple inhibition of endothelial NO synthesis. Our findings should contribute to a better understanding of the pathophysiological role of ADMA in arteriosclerosis.

Dexamethasone lacks effect on blood pressure in mice with a disrupted endothelial NO synthase gene

Cushing’s syndrome and systemic administration of glucocorticoids are associated with hypertension, but the underlying molecular mechanism is only partially understood. We have shown previously that dexamethasone downregulates the expression of the endothelial NO synthase (eNOS) gene in human endothelial cells and in the rat and that this may contribute to the blood pressure-raising effect of the steroid [Proc. Natl. Acad. Sci. USA 96 (1999) 13357]. In the current communication, we demonstrated that dexamethasone increased mean arterial blood pressure in wild-type C-57 Bl6 mice (eNOS +/+ mice), but had no effect on blood pressure in mice with a disrupted eNOS gene (eNOS −/− mice) derived from the same strain. The NOS inhibitor ethylisothiourea, used for control purposes, showed a hypertensive effect in eNOS +/+ mice, but no such effect in eNOS −/− mice. Serum NO 2 −/NO 3 − levels, an indicator of total body NO synthesis, decreased significantly when eNOS +/+ mice were treated with dexamethasone. eNOS −/− mice had lower serum NO 2 −/NO 3 − levels per se, which were not changed significantly by dexamethasone. Dexamethasone decreased the expression of eNOS in three major organs of the mouse investigated, namely the heart, the liver, and the kidney. We conclude that the expressional downregulation of eNOS and the ensuing reduction in vascular NO production contributes to the hypertension caused by glucocorticoids.

Effects of bulbus Fritillaria water extract on blood pressure and renal functions in the L-NAME-induced hypertensive rats

A pharmacological inhibition of nitric oxide synthase (NOS) in rats produces renal vasoconstriction, renal dysfunction, and hypertension. The present study was aimed at investigating whether Bulbus Fritillaria water extract (BFWE) ameliorates N G-nitro- l-arginine methylester (L-NAME)-induced hypertension. Treatment of rats with L-NAME (60 mg/l drinking water, 4 weeks) caused a sustained increase in systolic blood pressure (SBP). The NO concentration in plasma and NO productions in the vascular tissues of the L-NAME-treated group were significantly reduced as compared with those in the control, whereas the expressions of NOS proteins were not altered. BFWE restored SBP to normal level in the L-NAME-treated hypertensive rats. Moreover , BFWE was able to preserve the vascular NO production and plasma NO metabolites concentration without changes of the expression NOS proteins. The renal functional parameters including urinary volume, sodium excretion, and creatinine clearance (Ccr) were significantly restored in rats co-treated with BFWE and L-NAME compared to the L-NAME-treated group. Taken together, these results suggest that BFWE prevents the increase of SBP in the L-NAME-induced hypertension that may have been caused by enhanced generation of vascular NO and amelioration of renal functions.

Hemoglobin and red blood cells alter the response of expired nitric oxide to mechanical forces.

Expired nitric oxide (NO(e)) varies with hemodynamic or ventilatory perturbations, possibly due to shear stress- or stretch-stimulated NO production. Since hemoglobin (Hb) binds NO, NO(e) changes may reflect changes in blood volume and flow. To determine the role of blood and mechanical forces, we measured NO(e) in anesthetized rabbits, as well as rabbit lungs perfused with buffer, red blood cells (RBCs) or Hb following changes in flow, venous pressure (P(v)), and positive end-expiratory pressure (PEEP). In buffer-perfused lungs decreases in flow and P(v) reduced NO(e), but NO(e) rose when RBCs and Hb were present. These findings are consistent with changes in vascular NO production, whose detection is obscured in blood-perfused lungs by the more dominant effect of Hb NO scavenging. PEEP decreased NO(e) in all perfused lungs but increased NO(e) in live rabbits. The NO(e) fall with PEEP in isolated lungs is consistent with flow redistribution from alveolar septal capillaries to extra-alveolar vessels and decreased surface area or a direct, stretch-mediated depression of lung epithelial NO production. In live rabbits, increased NO(e) may reflect blood flow reduction and decreased Hb NO scavenging and/or autonomic responses that increase NO production. We conclude that blood and systemic responses render it difficult to use NO(e) changes as an accurate measure of lung tissue NO production.

Canine Coronary Microvessel NO Production Regulates Oxygen Consumption in ecNOS Knockout Mouse Heart

We have previously shown that NO production by tissues following stimulation with bradykinin or other agonists can regulate oxygen consumption in skeletal muscle, heart and kidney. From those studies and from those using agonists, which classically release NO from blood vessels and which are unable to regulate tissue oxygen consumption in heart from ecNOS knockout mice, we concluded that vascular NO production is capable of regulating tissue oxygen consumption. The goal of these studies was to directly address the concept that NO production by blood vessels can regulate tissue oxygen consumption using a classical transfer paradigm. Microvessels, capable of producing NO, were prepared from canine hearts using a sieving technique, cardiac tissue was taken from mice lacking the ability to produce NO from ecNOS (ecNOS −/− mice) and tissue oxygen consumption measured in vitro using a Clark type electrode in a sealed chamber. Bradykinin (10−7to 10−4M) had no effect on tissue oxygen consumption when administered to heart from ecNOS –/mice as expected and no effect on oxygen consumption by isolated canine coronary microvessels (0±5% at 10−5M). However when coronary microvessels were co-incubated with heart from ecNOS −/− mice, bradykinin caused a dose dependent reduction in tissue oxygen consumption reaching a maximum of 44±10% at 10−4M. The effects of bradykinin were entirely abolished by L -NAME. The calculated concentration range for NO in these studies was 2.9 to 293 n M, within estimated physiologic range for the activity of NO on cytochrome oxidase. These data indicate that coronary microvessels can regulate cardiac oxygen consumption through a NO dependent mechanism.

Limb blood flow during exercise is dependent on nitric oxide.

We have recently reported that hypercholesterolemia reduces aerobic exercise capacity in mice and that this is associated with a reduced endothelium-dependent vasodilator function, endothelium-derived nitric oxide (EDNO) production, and urinary nitrate excretion. These findings led us to test the hypothesis that EDNO production contributes significantly to limb blood flow during exercise and to determine whether loss of EDNO production is responsible for the decline in exercise capacity observed in hypercholesterolemia. Twelve-week-old wild-type (E+; n=9) and apoE-deficient (E-; n=9) C57BL/6J mice were treadmill-tested to measure indices defining exercise capacity on a metabolic chamber-enclosed treadmill capable of measuring oxygen uptake and carbon dioxide excretion. Urine was collected before and after treadmill exercise for determination of vascular NO production assessed by urinary nitrate excretion. The wild-type mice were then given nitro-L-arginine (E+LNA) in the drinking water (6 mg/dL) for 4 days before undergoing a second treadmill testing and urinary nitrate measurement. An additional set of 12-week-old wild-type mice was divided into 2 groups: 1 receiving regular water (E+; n=8) and 1 administered LNA for 4 days (E+LNA; n=8). These mice, along with an additional set of E mice (n=8), underwent treadmill testing to determine maximal oxygen uptake (VO2max). The mice were then cannulated such that the tip of the tubing was positioned in the ascending aorta. Fluorescent microspheres (20000) were infused into the carotid cannula while the mice were sedentary and again while approaching VO2max. When the mice were euthanized, the running muscles were collected and fluorescence intensity was measured to determine the peak-exercise redistribution of blood flow to the running muscles (expressed as percentage of total cardiac output, %COrm) during both states. Both E+LNA and E- mice demonstrated a markedly reduced postexercise urinary nitrate excretion, aerobic capacity, and %COrm at VO2max compared with E+. EDNO contributes significantly to limb blood flow during exercise. Conditions that reduce EDNO production disturb the hyperemic response to exercise, resulting in a reduced exercise capacity.

Neutral endopeptidase and angiotensin-converting enzyme inhibitors increase nitric oxide production in isolated canine coronary microvessels by a kinin-dependent mechanism.

Bradykinin is a substrate for both neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). Our previous studies showed that ACE inhibitors can stimulate nitric oxide production in coronary microvessels, which is mediated by local kinins. Whether inhibition of NEP also can affect local vascular NO production has not been established. To determine the role of NEP in the control of NO production, coronary microvessels were isolated from seven mongrel dogs. Two NEP inhibitors, phosphoramidon and thiorphan, and an ACE inhibitor, ramiprilat, were used. Nitrite, the metabolite of NO in aqueous solution, was measured by using the Griess reaction. Phosphoramidon and thiorphan (10(-6) M) increased nitrite production from 80 +/- 6 to 136 +/- 6 and 144 +/- 7 pmol/mg, respectively. Ramiprilat (10(-8) M) increased nitrite production from 78 +/- 6 to 155 +/- 7 pmol/mg wet weight. The effect of these agents on nitrite release was blocked by L-NAME, which inhibits NO synthase, HOE-140, which blocks bradykinin B2-receptor, and dichloroisocoumarin, which blocks kinin-forming enzymes. These results clearly indicate that inhibition of kinin metabolism by using neutral endopeptidase inhibitors increases NO production from coronary microvessels. Thus neutral endopeptidase plays an important role in local kinin-modulated NO production in the coronary microcirculation and NEP inhibitors may be useful clinical tools in treatment of cardiovascular disease.

Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels.

Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (chi 2(1) = 16.04, P = .000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NOx levels, and measured genotype analysis showed that plasma NOx levels in those homozygous for the rare allele (64.9 +/- 7.8 mumol/L) were significantly higher (P = .000242) than those homozygous for the common allele (30.2 +/- 3.1 mumol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NOx levels (P = .0066). While many environmental factors have been shown to alter transiently plasma NOx levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NOx, i.e. basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.