In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Enhanced vasoconstrictor responses in eNOS deficient mice.

Previous studies suggest that vasoconstriction is modulated by nitric oxide (NO). Contractions to ET-1 and/or thromboxane may be enhanced during chronic deficiency in expression or activity of NO synthase (NOS). Multiple isoforms of NOS are expressed within the vessel wall and purely pharmacological approaches cannot define the role of each. We tested the hypothesis that vasoconstriction to endothelin-1 (ET-1) and/or the thromboxane mimetic, U46619, is enhanced under conditions of chronic, selective deficiency in endothelial NOS (eNOS-/-) by examining responses in aorta from eNOS-/- mice compared to wild type (eNOS+/+). ET-1 produced dose-dependent contraction of aorta from eNOS+/+ mice that was increased twofold following acute inhibition of all NOS isoforms with N(G)-nitro-L-arginine (L-NNA). In eNOS-/- mice, contractions to ET-1 were increased twofold compared to eNOS+/+. L-NNA had no effect. Although contraction of the aorta to thromboxane mimetic U46619 was increased at lower concentrations, maximal contractions to U46619 were not increased following acute inhibition of NOS or in eNOS-/- mice. These studies provide direct evidence that vasoconstriction to ET-1 and thromboxane is augmented in the face of eNOS deficiency, demonstrating that eNOS normally inhibits vascular contractile responses.     

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.     

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.     

Role of eNOS-derived NO in the postischemic anti-inflammatory effects of antecedent ethanol ingestion in murine small intestine

Ingestion of low levels of ethanol 24 h before [ethanol preconditioning (EPC)] ischemia and reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and adhesion (LA), effects that were abolished by adenosine A(2) receptor (ADO-A(2)R) antagonists or nitric oxide (NO) synthase (NOS) inhibitors. The aims of this study were to determine whether NO derived from endothelial NOS (eNOS) during the period of ethanol exposure triggered entrance into this preconditioned state and whether these events were initiated by an ADO-A(2)R-dependent mechanism. Ethanol or distilled water vehicle was administered to C57BL/6J [wild type (WT)] or eNOS-deficient (eNOS-/-) mice by gavage. Twenty-four hours later, the superior mesenteric artery was occluded for 45 min. LR and LA were quantified by intravital microscopy after 30 and 60 min of reperfusion. I/R increased LR and LA in WT mice, effects that were abolished by EPC or NO donor preconditioning (NO-PC). NO-PC was not attenuated by coincident administration of an ADO-A(2)R antagonist. I/R increased LR and LA in eNOS-/- mice to levels comparable with those noted in WT animals. However, EPC only slightly attenuated postischemic LR and LA, whereas NO-PC remained effective as a preconditioning stimulus in eNOS-/- mice. Preconditioning with an ADO-A(2)R agonist (which we previously demonstrated prevents I/R-induced LR and LA in WT animals) failed to attenuate these postischemic adhesive responses in eNOS-/- mice. Our results indicate that EPC is triggered by NO formed secondary to ADO-A(2)R-dependent eNOS activation during the period of ethanol exposure 24 h before I/R.     

Exercise training normalizes impaired NOS-dependent responses of cerebral arterioles in type 1 diabetic rats

Our goal was to examine whether exercise training (ExT) could normalize impaired nitric oxide synthase (NOS)-dependent dilation of cerebral (pial) arterioles during type 1 diabetes (T1D). We measured the in vivo diameter of pial arterioles in sedentary and exercised nondiabetic and diabetic rats in response to an endothelial NOS (eNOS)-dependent (ADP), an neuronal NOS (nNOS)-dependent [N-methyl-D-aspartate (NMDA)], and a NOS-independent (nitroglycerin) agonist. In addition, we measured superoxide anion levels in brain tissue under basal conditions in sedentary and exercised nondiabetic and diabetic rats. Furthermore, we used Western blot analysis to determine eNOS and nNOS protein levels in cerebral vessels/brain tissue in sedentary and exercised nondiabetic and diabetic rats. We found that ADP and NMDA produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic rats. In contrast, ADP and NMDA produced only minimal vasodilation in sedentary diabetic rats. ExT restored impaired ADP- and NMDA-induced vasodilation observed in diabetic rats to that observed in nondiabetics. Nitroglycerin produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic and diabetic rats. Superoxide levels in cortex tissue were similar in sedentary and exercised nondiabetic rats, were increased in sedentary diabetic rats, and were normalized by ExT in diabetic rats. Finally, we found that eNOS protein was increased in diabetic rats and further increased by ExT and that nNOS protein was not influenced by T1D but was increased by ExT. We conclude that ExT can alleviate impaired eNOS- and nNOS-dependent responses of pial arterioles during T1D.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

Pioglitazone protects the myocardium against ischemia-reperfusion injury in eNOS and iNOS knockout mice

Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1alpha levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS-/- and iNOS-/- mice. Male C57BL/6 wild-type (WT), eNOS-/-, and iNOS-/- mice received 10 mg.kg(-1).day(-1) Pio (Pio+) or water alone (Pio-) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4+/-1.4% vs. 39.0+/-1.1%; P<0.001), as well as in the eNOS-/- (32.0+/-1.6% vs. 44.2+/-1.9%; P<0.001) and iNOS-/- (18.0+/-1.2% vs. 45.5+/-2.3%; P<0.001) mice. The protective effect of Pio in eNOS-/- mice was smaller than in the WT (P<0.001) and iNOS-/- (P<0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS-/- mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS-/- and iNOS-/-, mice. Pio increased the myocardial 6-keto-PGF1alpha levels and cPLA2 and COX2 activity in the WT, eNOS-/-, and iNOS-/- mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.     

Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice

One of the main factors that control vasoreactivity and angiogenesis is nitric oxide produced by endothelial nitric oxide synthase (eNOS). We recently showed that knocking out eNOS induces an important reduction of mitochondrial oxidative capacity in slow-twitch skeletal muscle. Here we investigated eNOS's role in physical activity and contribution to adaptation of muscle energy metabolism to exercise conditions. Physical capacity of mice null for the eNOS isoform (eNOS-/-) was estimated for 8 wk with a voluntary wheel-running protocol. In parallel, we studied energy metabolism enzyme profiles and their response to voluntary exercise in cardiac and slow-twitch soleus (Sol) and fast-twitch gastrocnemius (Gast) skeletal muscles. Weekly averaged running distance was two times lower for eNOS-/- (4.09 +/- 0.42 km/day) than for wild-type (WT; 7.74 +/- 0.42 km/day; P < 0.01) mice. Average maximal speed of running was also lower in eNOS-/- (17.2 +/- 1.4 m/min) than WT (21.2 +/- 0.9 m/min; P < 0.01) mice. Voluntary exercise influenced adaptation to exercise specifically in Sol muscle. Physical activity significantly increased Sol weight by 22% (P < 0.05) in WT but not eNOS-/- mice. WT Sol muscle did not change its metabolic profile in response to exercise, in contrast to eNOS-/- muscle, in which physical activity decreased cytochrome-c oxidase (COX; -36%; P < 0.05), citrate synthase (-37%; P < 0.06), and creatine kinase (-24%, P < 0.01) activities. Voluntary exercise did not change energy enzyme profile in heart (except for 39% increase in COX activity in WT) or Gast muscle. These results suggest that eNOS is necessary for maintaining a suitable physical capacity and that when eNOS is downregulated, even moderate exercise could worsen energy metabolism specifically in oxidative skeletal muscle.     

Modulation of mouse cardiac function in vivo by eNOS and ANP

To study the role of endothelial nitric oxide synthase (eNOS) in cardiac function, we compared eNOS expression, contractility, and relaxation in the left ventricles of wild-type and eNOS-deficient mice. eNOS immunostaining is localized to the macro- and microvascular endothelium throughout the myocardium in wild-type mice and is absent in eNOS-/- mice. Whereas blood pressure is elevated in eNOS-/- mice, baseline cardiac contractility (dP/dt(max)) is similar in wild-type and eNOS-/- mice (9,673 +/- 2, 447 and 9,928 +/- 1,566 mmHg/s, respectively). The beta-adrenergic agonist isoproterenol (Iso) at doses of >/=1 ng causes enhanced increases in dP/dt(max) in eNOS-/- mice compared with wild-type controls in vivo (P < 0.01) as well as in Langendorff isolated heart preparations (P < 0.02). beta-Adrenergic receptor binding (B(max)) is not significantly different in the two groups of animals (B(max) = 41.4 +/- 9.4 and 36.1 +/- 5.1 fmol/mg for wild-type and eNOS-/-). Iso-stimulated ventricular relaxation is also enhanced in the eNOS-/- mice, as measured by dP/dt(min) in the isolated heart. However, baseline ventricular relaxation is normal in eNOS-/- mice (tau = 5.2 +/- 1.0 and 5.6 +/- 1.5 ms for wild-type and eNOS-/-, respectively), whereas it is impaired in wild-type mice after NOS inhibition (tau = 8.3 +/- 2.4 ms). cGMP levels in the left ventricle are unaffected by eNOS gene deletion (wild-type: 3.1 +/- 0.8 pmol/mg, eNOS-/-: 3.1 +/- 0.6 pmol/mg), leading us to examine the level of another physiological regulator of cGMP. Atrial natriuretic peptide (ANP) expression is markedly upregulated in the eNOS-/- mice, and exogenous ANP restores ventricular relaxation in wild-type mice treated with NOS inhibitors. These results suggest that eNOS attenuates both inotropic and lusitropic responses to beta-adrenergic stimulation, and it also appears to regulate baseline ventricular relaxation in conjunction with ANP.     

Maternal aggression in endothelial nitric oxide synthase-deficient mice

Lactating female rodents protect their pups by expressing fierce aggression, termed maternal aggression, toward intruders. Mice lacking the neuronal nitric oxide synthase gene (nNOS-/-) exhibit significantly impaired maternal aggression, but increased male aggression, suggesting that nitric oxide (NO) produced by nNOS has opposite actions in maternal and male aggression. In contrast, mice lacking the endothelial nitric oxide synthase gene (eNOS-/-) exhibit almost no male aggression, suggesting that NO produced by eNOS facilitates male aggression. In the present study, maternal aggression in eNOS-/- mice was examined and found to be normal relative to wild-type (WT) mice in terms of the percentage displaying aggression, the average number of attacks against a male intruder, and the total amount of time spent attacking the male intruder. The eNOS-/- females also displayed normal pup retrieval behavior. Because a significant elevation of citrulline, an indirect marker of NO synthesis, occurs in neurons of the hypothalamus of lactating WT mice in association with maternal aggression, we examined the brains of eNOS-/- females for citrulline immunoreactivity following an aggressive encounter. The aggressive eNOS-/- females exhibited a significant elevation of citrulline in the medial preoptic nucleus and the subparaventricular zone of the hypothalamus relative to unstimulated lactating eNOS-/- females. Taken together, these results suggest that NO produced by eNOS neither facilitates nor inhibits maternal aggression and that NO produced by eNOS has a different role in maternal and male aggression.     

Advancing age produces sex differences in vasomotor kinetics during and after skeletal muscle contraction

Little is known of the vasomotor responses of skeletal muscle arterioles during and following muscle contraction. We hypothesized that aging leads to impaired arteriolar responses to muscle contraction and recovery. Nitric oxide (NO) availability, which is age dependent, has been implicated in components of these kinetics. Therefore, we also hypothesized that changes in the kinetics of vascular responses are associated with the NO pathway. Groups were young (3 mo), old (24 mo), endothelial NO synthase knockout (eNOS-/-), and N(G)-nitro-L-arginine (L-NA)-treated male and female C57BL/6 mice. The kinetics of vasodilation during and following 1 min of contractions of the gluteus maximus muscle were recorded in second-order (regional distribution) and third-order (local control) arterioles. Baseline, peak (during contraction), and maximal diameters (pharmacological) were not affected by age or sex. The kinetics of dilation and recovery were not different between males and females at the young age. There was a significant slowing of vasodilation at the onset of contractions (approximately 2-fold; P < 0.05) and a significant speeding of recovery ( approximately 5-fold; P < 0.05) in old males vs. old females and vs. young eNOS-/-, and L-NA did not affect the kinetics at the onset of muscle contraction. eNOS-/- mimicked the rapid recovery of old males in second-order arterioles; acute NO production (L-NA) explained approximately 50% of this effect. These data demonstrate fundamental age-related differences between the sexes in the dynamic function of skeletal muscle arterioles. Understanding how youthful function persists in females but not males may provide therapeutic insight into clinical interventions to maintain dynamic microvascular control of nutrient supply with age.     

Chronic resveratrol treatment restores vascular responsiveness of cerebral arterioles in type 1 diabetic rats

Decreased dilation of cerebral arterioles via an increase in oxidative stress may be a contributing factor in the pathogenesis of diabetes-induced complications leading to cognitive dysfunction and/or stroke. Our goal was to determine whether resveratrol, a polyphenolic compound present in red wine, has a protective effect on cerebral arterioles during type 1 diabetes (T1D). We measured the responses of cerebral arterioles in untreated and resveratrol-treated (10 mg·kg(-1)·day(-1)) nondiabetic and diabetic rats to endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase (NOS)-dependent agonists and to a NOS-independent agonist. In addition, we harvested brain tissue from nondiabetic and diabetic rats to measure levels of superoxide under basal conditions. Furthermore, we used Western blot analysis to determine the protein expression of eNOS, nNOS, SOD-1, and SOD-2 in cerebral arterioles and/or brain tissue from untreated and resveratrol-treated nondiabetic and diabetic rats. We found that T1D impaired eNOS- and nNOS-dependent reactivity of cerebral arterioles but did not alter NOS-independent vasodilation. While resveratrol did not alter responses in nondiabetic rats, resveratrol prevented T1D-induced impairment in eNOS- and nNOS-dependent vasodilation. In addition, superoxide levels were higher in brain tissue from diabetic rats and resveratrol reversed this increase. Furthermore, eNOS and nNOS protein were increased in diabetic rats and resveratrol produced a further increased eNOS and nNOS proteins. SOD-1 and SOD-2 proteins were not altered by T1D, but resveratrol treatment produced a decrease in SOD-2 protein. Our findings suggest that resveratrol restores vascular function and oxidative stress in T1D. We suggest that our findings may implicate an important therapeutic potential for resveratrol in treating T1D-induced cerebrovascular dysfunction.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection

The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.     

ADP-induced pial arteriolar dilation in ovariectomized rats involves gap junctional communication

It was previously shown that, despite the loss of nitric oxide (NO) dependence, ADP-induced pial arteriolar dilation was not attenuated in estrogen-depleted [i.e., ovariectomized (Ovx)] rats. Additional evidence suggested that the NO was replaced by an endothelium-dependent hyperpolarizing factor (EDHF)-like mechanism. To further characterize the nascent EDHF role in Ovx females, the current study was undertaken to test whether, in Ovx rats, ADP-induced pial arteriolar dilation retained its endothelial dependence and whether gap junctions are involved in that response. A closed cranial window and intravital microscopy system was used to monitor pial arteriolar diameter changes in anesthetized rats. The endothelial portion of the ADP-induced dilation was evaluated using light dye endothelial injury (L/D). The study was organized around three experimental approaches. First, the responses of pial arterioles to ADP before and after L/D exposure in intact and Ovx female rats were tested. L/D reduced the ADP response by 50-70% in both groups, thereby indicating that the endothelium dependence of ADP-induced vasodilation is not altered by chronic estrogen depletion. Second, the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) and the prostanoid synthesis inhibitor indomethacin (Indo) were coapplied. In intact females, L-NNA-Indo attenuated the response to ADP by 50%, with no further changes upon the addition of L/D. On the other hand, L-NNA-Indo did not affect ADP reactivity in Ovx rats, but subsequent L/D exposure reduced the ADP response by >50%. The NO-prostanoid-independent, but endothelium-dependent, nature of the response in Ovx females is a hallmark of EDHF participation. Third, gap junctional inhibition strategies were applied. A selective inhibitor of gap junctional function, Gap 27, did not affect ADP reactivity in intact females but reduced the the ADP response by 50% in Ovx females. A similar result was obtained following application of a connexin43 antisense oligonucleotide. These findings suggest that the nascent EDHF dependency of ADP-induced pial arteriolar dilation in Ovx females involves connexin43-related gap junctional communication.     

Distinct modulation of the endothelin-1 pathway in iNOS-/- and eNOS-/- mice

We hypothesized that constitutive endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) have opposite effects on the regulation of endothelin and its receptors. We therefore sought to determine whether deletions of iNOS or eNOS genes in mice modulate pressor responses to endothelin and the expression of ETA and ETB receptors in a similar fashion. Despite unchanged baseline hemodynamic parameters, anesthetized iNOS-/- mice displayed reduced pressor responses to endothelin-1, but not to that of IRL-1620, a selective ETB agonist. Protein content of cardiac ETA receptors was reduced in iNOS-/- mice compared with wild-type mice, but that of ETB receptors was unchanged. Anesthetized eNOS-/- mice presented a hypertensive state, accompanied by an enhanced pressor response to intravenous endothelin-1, whereas the pressor response to IRL-1620 was reduced. Protein levels were also found to be increased for ETA receptors, but reduced for ETB receptors, in cardiac tissues of eNOS-/- mice. In conscious animals, both strains responded equally to the hypotensive effect of an ETA antagonist, ABT-627, whereas orally administered A-192621, an ETB antagonist, increased MAP to a greater extent in eNOS-/- than in wild-type mice. Furthermore, significant levels of immunoreactive endothelin were found in mesenteric arteries in eNOS-/- but not in iNOS-/- or wild-type congeners. Our study shows that repression of iNOS or eNOS has differential effects on endothelin-1 and its receptors. We have also shown that the heart is the main organ in which iNOS or eNOS repression induces important alterations in protein content of endothelin receptors in adult mice.     

Endothelial nitric oxide is not involved in circadian rhythm generation of blood pressure: experiments in wild-type C57 and eNOS knock-out mice under light-dark and free-run conditions

Endothelial nitric oxide synthase knock out mice (eNOS-/-) are mildly hypertensive in comparison to wild-type (WT) mice. Hypertension in eNOS-/- mice is partly the result of an increase in peripheral resistance due to the absence of the vasodilatory action of NO. No data are available for these animals regarding the 24 h blood pressure profile under the 12:12 h light-dark cycle (LD) and constant dark (DD) conditions. Therefore, this study aimed to investigate by radiotelemetry the circadian rhythms in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of six eNOS-/- mice and five wild-type mice under LD and DD. Data were collected beginning 3 wks after operation (implantation of sensor) for 2 wks under LD and for another 2 wks thereafter under DD. Our results show that eNOS-/- mice were hypertensive under all experimental conditions. SBP and DBP were significantly higher by about 15% in eNOS-/- mice. No differences were found in the pattern of the circadian rhythms, rhythmicity, or period lengths during LD or DD. The genetic deletion of eNOS seems to lead to higher SBP and DBP, but the circadian blood pressure pattern is still preserved with higher values during the night (active phase) and lower values during the daytime (rest phase). Thus, endothelial-derived NO plays an important role in the regulation of vascular tone and haemodynamics, but it is not important for the circadian organization of SBP and DBP.     

Effect of NO synthase inhibition on cardiovascular circadian rhythms in wild-type and eNOS-knock-out mice

Cardiovascular functions (blood pressure [BP], heart rate [HR]) were radiotelemetrically studied in endothelial nitric oxide synthase (NOS) knock-out mice (eNOS-/-) and their wild type C57BL/6 (WT) controls. Studies were performed with and without inhibition of the NOS with the non-specific inhibitor N(omega)-Nitro-L-Arginin-Methylester (L-NAME). Six eNOS-/-and five WT mice, kept under a light:dark schedule of 12:12 h (lights on 07:00 h), were treated with L-NAME in tap water containing different concentrations (94, 282, and 940 mg/kg) each for three days. Under control conditions, the eNOS-/-mice are mildly hypertensive in comparison to WT. L-NAME increased systolic [SBP] and diastolic [DBP] blood pressures in WT mice to the levels of eNOS-/-mice after two days of L-NAME application with no dose-dependency, whereas L-NAME had no effects on SBP and DBP in eNOS-/-mice. In neither mouse strain were the circadian rhythms in BP and HR affected by drug treatment. The similarity of the 24 h BP profiles in eNOS-/-and L-NAME-treated WT mice support the notion that only the enothelial NOS and not other NOS isoenzymes are of importance for hypertension in the knock-out mouse strain.     

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.