Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase

Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis. (Mol Cell Biochem 264: 193–200, 2004)     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis.     

Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9–12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre‐deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non‐functional eNOS allele through Cre recombination. This mouse will allow time‐ and cell‐specific eNOS deletion. genesis 50:685–692, 2012. © 2012 Wiley Periodicals, Inc.     

Reproductive function in female mice lacking the gene for endothelial nitric oxide synthase.

Nitric oxide (NO) acts as a neuronal messenger in both the central and peripheral nervous systems and has been implicated in reproductive physiology and behavior. Pharmacological inhibition of nitric oxide synthase (NOS) with the nonspecific NOS inhibitor, l-N(G)-nitro-Arg-methyl ester (l-NAME), induced deficits in both the number of ovarian rupture sites and the number of oocytes recovered in the oviducts of mice. Female neuronal NOS knockout (nNOS-/-) mice have normal numbers of rupture sites, but reduced numbers of oocytes recovered following systemic injections of gonadotropins, suggesting that NO produced by nNOS accounts, in part, for deficits in ovulatory efficiency observed after l-NAME administration. Additionally, endothelial NOS knockout (eNOS-/-) mice have reduced numbers of ovulated oocytes after superovulation. Because endothelial NOS has been identified in ovarian follicles, and because of the noted reduced breeding efficiency of eNOS-/- mice, the present study sought to determine the role of NO from eNOS in mediating the number of rupture sites present after ovulation. Estrous cycle length and variability were consistently reduced in eNOS-/- females. The number of rupture sites was normal in eNOS-/- mice under natural conditions and after administration of exogenous GnRH. After exogenous gonadotropin administration, eNOS-/- females displayed a significant reduction in the number of ovarian rupture sites. Female eNOS-/- mice also produced fewer pups/litter compared to WT mice. These data suggest that NO from endothelial sources might play a role in mediating rodent ovulation and may be involved in regulation of the timing of the estrous cycle.     

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

Whereas altered nitric oxide (NO.) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO. formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO. synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO. formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by the L-[(14)C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO. generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO. synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.     

Plasma membrane-associated endothelial nitric oxide synthase and activity in aging rat aortic vascular endothelia markedly decline with age

The mechanisms leading to the age-related loss of endothelial nitric oxide (NO) and NO-dependent vasodilation remain largely unknown. Freshly isolated endothelium from young (6 months) and old (36 months) F344xBrN rats were analyzed for endothelial nitric oxide synthase (eNOS) protein, its subcellular distribution, and association with regulatory proteins. Results show that both vessel ring vasoreactivity and A23187-induced eNOS activity in isolated endothelial cells significantly (p < or = 0.05) declined with age. Levels of cGMP, a reliable marker for NO bioactivity also declined significantly (p < or = 0.01). However, no change in overall eNOS protein was evident. Subcellular fractionation studies revealed an age-related loss in active, plasma membrane-bound eNOS relative to eNOS in the Golgi/cytosol of the endothelium. Plasma membrane-associated eNOS in aged endothelium was also less complexed with the activating proteins Hsp90 and Akt and more associated with to caveolin-1, which inhibits eNOS activity. These results suggest that age-dependent loss of NO may be partly caused by differences in eNOS subcellular distribution and its association with inhibitory proteins.     

Increasing dihydrobiopterin causes dysfunction of endothelial nitric oxide synthase in rats in vivo

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.     

Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.     

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.     

Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction

Superoxide, which can limit nitric oxide bioavailability, has been implicated in blood cell-vessel wall interactions observed in sickle cell transgenic (beta(S)) mice. Here we report that nonselective chemical inhibition of nitric oxide synthase isoforms dramatically reduces the enhanced leukocyte and platelet adhesion normally observed in cerebral venules of beta(S) mice. Although genetic deficiency of vascular wall inducible nitric oxide synthase does not alter adhesion responses in beta(S) mice, a significant attenuation is noted in beta(S) mice with vascular wall endothelial nitric oxide synthase (eNOS) deficiency, while the adhesion responses are exacerbated when eNOS is overexpressed in microvessels. The eNOS-mediated enhancement of blood cell adhesion is reversible by pretreatment with sepiapterin (which generates the eNOS cofactor tetrahydrobiopterin) or polyethyleneglycol-superoxide dismutase, implicating a role for eNOS-dependent superoxide production. These findings suggest that an imbalance between eNOS-derived nitric oxide and superoxide, both generated by the vessel wall, is critical to the proinflammatory and prothrombogenic phenotype that is assumed by the microvasculature in sickle cell disease.     

Crystallographic studies on endothelial nitric oxide synthase complexed with nitric oxide and mechanism-based inhibitors

The crystal structure of the endothelial nitric oxide synthase (NOS) heme domain complexed with NO reveals close hydrogen bonding interactions between NO and the terminal guanidino nitrogen of the substrate, L-arginine. Dioxygen is expected to bind in a similar mode which will facilitate proton abstraction from L-Arg to dioxygen, a required step for O-O bond cleavage. Structures of mechanism-based NOS inhibitors, N(5)-(1-iminoethyl)-L-ornithine and N-(3-(aminomethyl)benzyl)acetamidine, provide clues on how this class of compounds operate as suicide substrate inhibitors leading to heme oxidation.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Anthocyanins attenuate endothelial dysfunction through regulation of uncoupling of nitric oxide synthase in aged rats

Endothelial dysfunction is one of the main age‐related arterial phenotypes responsible for cardiovascular disease (CVD) in older adults. This endothelial dysfunction results from decreased bioavailability of nitric oxide (NO) arising downstream of endothelial oxidative stress. In this study, we investigated the protective effect of anthocyanins and the underlying mechanism in rat thoracic aorta and human vascular endothelial cells in aging models. In vitro, cyanidin‐3‐rutinoside (C‐3‐R) and cyanidin‐3‐glucoside (C‐3‐G) inhibited the d‐galactose (d‐gal)‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p21, and p16^INK4a. Anthocyanins blocked d‐gal‐induced reactive oxygen species (ROS) formation and NADPH oxidase activity. Anthocyanins reversed d‐gal‐mediated inhibition of endothelial nitric oxide synthase (eNOS) serine phosphorylation and SIRT1 expression, recovering NO level in endothelial cells. Also, SIRT1‐mediated eNOS deacetylation was shown to be involved in anthocyanin‐enhanced eNOS activity. In vivo, anthocyanin‐rich mulberry extract was administered to aging rats for 8 weeks. In vivo, mulberry extract alleviated endothelial senescence and oxidative stress in the aorta of aging rats. Consistently, mulberry extract also raised serum NO levels, increased phosphorylation of eNOS, increased SIRT1 expression, and reduced nitrotyrosine in aortas. The eNOS acetylation was higher in the aging group and was restored by mulberry extract treatment. Similarly, SIRT1 level associated with eNOS decreased in the aging group and was restored in aging plus mulberry group. These findings indicate that anthocyanins protect against endothelial senescence through enhanced NO bioavailability by regulating ROS formation and reducing eNOS uncoupling.     

Involvement of vascular endothelial nitric oxide synthase in development of experimental diabetic nephropathy in rats

Endothelial nitric-oxide synthase (eNOS) acts as a common pathogenic pathway in diabetic nephropathy (DN). However, its functional consequences are still not fully understood. Caveolin, a membrane protein, inhibits the eNOS by making caveolin-eNOS complex, and its expression is upregulated during diabetes mellitus (DM). This study was designed to determine the role of caveolin in eNOS-mediated NO synthesis and release in DN. DM in rat was induced by feeding of high-fat diet (HFD) for 2 weeks, followed by single dose of streptozotocin (STZ) (35 mg/kg, ip) further followed by HFD for further 8 weeks. Serum nitrite/nitrate ratio was measured to determine the plasma level of NO. Diabetic rat, after 6 weeks of STZ, developed elevated level of BUN, protein in urine, urinary output, serum creatinine, serum cholesterol, kidney weight, kidney weight/body weight, and renal cortical collagen content, while serum nitrite/nitrate concentration was significantly decreased as compared to normal control group. Treatment with sodium nitrite (NO donor), L: -arginine (NO precursor), daidzein (caveolin inhibitor), and combination of L: -arginine and daidzein for 2 weeks markedly attenuated these changes and increased serum nitrite/nitrate ratio. However, treatment with L-NAME, a eNOS inhibitor, significantly attenuated the L: -arginine-, daidzein-, or combination of L: -arginine and daidzein-induced ameliorative effects in DN. The finding of this study suggests that caveolin plays a vital role in the eNOS-mediated decrease in renal level of NO, which may be responsible for the development of DN in rats.     

Circadian organization in male mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-)

Circadian (approximately 24 h) rhythms in physiology and behavior are generated by the bilateral suprachiasmatic nucleus (SCN) of the anterior hypothalamus. For these endogenous rhythms to be synchronized with the external environment, light information must be transmitted to pacemaker cells within the SCN. This transmission of light information is accomplished via a direct retino-hypothalamic tract (RHT). Nitric oxide (NO), an endogenous gas that functions as a neurotransmitter, has been implicated as a messenger necessary for photic entrainment. Three isoforms of the enzyme that form NO, NO synthase, have been identified (a) in neurons (nNOS), (b) in the endothelial lining of blood vessels (eNOS), and (c) as an inducible form in macrophages (iNOS). The present study was undertaken to determine the specific role of eNOS in circadian organization and photic entrainment. Wild-type (WT) and eNOS-/- mice were initially entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was phase advanced. After an additional 3 weeks, animals were held in constant darkness (DD). eNOS-/- animals did not exhibit a deficit in the ability to entrain to the LD cycle, phase-shift locomotor activity, or free-run in constant conditions. Animals held in DD were killed after light exposure during either the subjective day or the subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos protein expression in the SCN of both WT and eNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that endothelial isoform of NOS may not be necessary for photic entrainment in mice.     

Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform of nitric oxide synthase

The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(-/-)], or nNOS gene [nNOS(-/-)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at 10(-8) and 10(-6) mol/l reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). N(G)-nitro-L-arginine methyl ester (L-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at 10(-8) and 10(-6) mol/l. The response to angiotensin II was enhanced in nNOS(-/-) mice (41% and 25% at 10(-8) and 10(-6) mol/l) and even more enhanced in eNOS(-/-) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). L-NAME led to complete constriction of Af in these groups. Media-to-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types 1A and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation.     

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.