Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Decreased viability of nitric oxide synthase double knockout mice

Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.     

Aged endothelial nitric oxide synthase knockout mice exhibit higher mortality concomitant with impaired open-field habituation and alterations in forebrain neurotransmitter levels

Endothelial nitric oxide synthase (eNOS) has been implicated in various brain and peripheral pathologies such as renal failure, heart failure or stroke. Consequently, the mortality rate of aged eNOS knockout mice (eNOS^–/–) was higher than that of age-matched (18–22 months old) controls. Only seven of the original 14 eNOS^–/– animals that participated in the study reached the age of 18 months or older, whereas no control mice died during this life span. In order to assess the behavioral and neurochemical consequences of chronic eNOS deficiency we examined whether the surviving aged eNOS^–/– mice showed changes in terms of motor, emotional, exploratory and neurochemical parameters. Aged eNOS^–/– mice showed reduced exploratory activity in the open-field with no habituation observable neither within sessions nor after repeated exposures. Pole test performance of eNOS^–/– mice was comparable to controls. In the elevated plus-maze eNOS^–/– mice did not differ from controls in terms of time spent in and entries into arms, but showed less locomotion on the open arms. The most prominent neurochemical alterations in the forebrains of aged eNOS^–/– mice were: (a) increased acetylcholine levels in the neostriatum; (b) decreased noradrenaline concentrations in the ventral striatum; and (c) lower serotonin levels in the frontal cortex and ventral striatum. The present findings suggest that mice which survived chronic eNOS-deficiency into old age, show some behavioral and neurochemical phenotypes distinct from adult eNOS^–/– mice.     

Expression of endothelial nitric oxide synthase is suppressed in the renal vasculature of angiotensinogen-gene knockout mice

We have attempted to elucidate the mechanism by which endothelial-type nitric oxide synthase (eNOS) is regulated in the kidney, with special reference to the role of renal hemodynamics and angiotensin II (Ang II). We compared angiotensinogen gene knockout (Atg−/−) mice, which lacked Ang II (resulting in sodium/water depletion and severe hypotension), with wild-type (Atg+/+) mice. Using Western blot analysis and the NADPH diaphorase histochemical reaction, we found that the expression and activity of eNOS were markedly lower in the renal vessels of Atg−/− mice compared with wild-type (Atg+/+) mice. Dietary salt loading significantly enhanced renal eNOS levels and increased blood pressure in Atg−/− mice, but severe hypotension almost abolished the effects of salt loading. In contrast, in Atg+/+ mice, altered salt intake or hydralazine had no effect on renal eNOS levels. These results suggest that perfusion pressure plays an essential role in maintaining renal vascular eNOS activity, whereas Ang II plays a supportive role, especially when renal circulation is impaired. This study was supported by Grants-in-Aid for Scientific Resarch 2001–2003, Japan Society for Promotion of Science (grant no. 13670735).     

Scrapie-infected mice and PrP knockout mice share abnormal localization and activity of neuronal nitric oxide synthase

PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains.     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Vasomotor control in mice overexpressing human endothelial nitric oxide synthase

Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.     

Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells.

Hyperglycemia is considered a primary cause of diabetic vascular complications. A hallmark of vascular disease is endothelial cell dysfunction characterized by diminished nitric-oxide (NO)-dependent phenomena such as vasodilation, angiogenesis, and vascular maintenance. This study was designed to investigate the effects of a high level of D-glucose on endothelial NO response, oxidative stress, and glucose metabolism. Bovine aortic endothelial cells (BAECs) were pretreated with a high concentration of glucose (HG) (22 mmol/L) for at least 2 weeks and compared with control cells exposed to 5 mmol/L glucose (NG). The effect of chronic hyperglycemia on endothelial NO-synthase (eNOS) activity and expression, glycogen synthase (GS) activity, extracellular-signal-regulated kinase (ERK 1,2), p38, Akt expression, and Cu/Zn superoxide-dismutse (SOD-1) activity and expression were determined. Western blot analysis showed that eNOS protein expression decreased in HG cells and was accompanied by diminished eNOS activity. The activity of GS was also significantly lower in the HG cells than in NG cells, 25.0+/-17.4 and 89+/-22.5 nmol UDP-glucose.mg protein(-1)x min(-1), respectively. Western blot analysis revealed a 40-60% decrease in ERK 1,2 and p38 protein levels, small modification of phosphorylated Akt expression, and a 30% increase in SOD-1 protein expression in HG cells. Although SOD expression was increased, no change was observed in SOD activity. These results support the findings that vascular dysfunction due to exposure to pathologically high D-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.     

Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis

In skeletal muscles, angiogenesis can be induced by increases in wall shear stress. To identify molecules involved in the angiogenic process, a method based on the use of BS-1 lectin-coated magnetic beads was developed to isolate a cellular fraction enriched in microvascular endothelial cells which are directly exposed to wall shear stress. Using such cellular fractions from skeletal muscles of C57 mice in which angiogenesis was induced by administration with the alpha(1)-adrenergic antagonist prazosin, we found the concentration of vascular endothelial growth factor (VEGF) increased in correlation to the duration of the prazosin stimulus. In contrast, the angiopoietin-2/tie-2 system was not changed even after 4days of prazosin treatment. In neuronal nitric oxide synthase (nNOS) knockout mice, the VEGF concentration was also elevated after prazosin treatment but remained almost unchanged in endothelial nitric oxide synthase (eNOS) knockout mice. However, eNOS (and not nNOS) knockout mice expressed higher levels of VEGF under non-stimulated conditions as compared to C57 mice. These results suggest that VEGF produced in endothelial cells is involved in angiogenesis in skeletal muscles of mice responding to the administration of systemic vasodilators. NO derived from eNOS and nNOS may be an important regulator of the angiogenic response in skeletal muscles in vivo.     

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test.Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05).Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.     

Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory process of certain major cardiac disorders including myocardial infarction and allograft rejection. However, the role of iNOS in acute myocardial ischemia has not been well defined. We determined the effects of genetically disruption of the intact iNOS system on cardiac tolerance to ischemia/reperfusion injury. Adult male wild-type (WT) and iNOS knockout (KO) B6,129 mice were subjected to 20 min global ischemia and 30 min reperfusion in a Langendorff isolated perfused heart model (37 degrees C, n = 10/each group). Ventricular contractile function, heart rate, coronary flow, and leakage of intracellular enzymes (CK and LDH) were not significantly different between the groups during pre-ischemia as well as reperfusion period (P > 0.05). Myocardial infarct size was also not significantly different between WT (20.2+/-2.0% of risk area) and KO mice (23.5+/-3.8%; Mean+/-SEM, P > 0.05). However, the post-ischemic heart rate was significantly preserved in KO as compared to WT (P < 0.05). We conclude that disruption of iNOS gene does not exacerbate ischemia/ reperfusion injury in the heart.     

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.     

Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) participates in many pathological events, and selective inhibition of iNOS has been shown to reduce ischemia-reperfusion (I/R) injury in different tissues. To further confirm its role in this injury process, I/R injury was observed in denervated cremaster muscles of iNOS-deficient (iNOS-/-) and wild-type mice. After 3-h ischemia and 90-min reperfusion, blood flow in reperfused muscle was 80 +/- 8.5% (mean +/- SE) of baseline at 10-min reperfusion and completely returned to the preischemia baseline after 20 min in iNOS-/- mice. In contrast, blood flow was 32 +/- 7.4% at 10 min and increased to 60 +/- 20% of the baseline level at 90 min in wild-type mice (P < 0.001 vs. iNOS-/- mice at all time points). The increased muscle blood flow in iNOS-/- mice was associated with significantly less vasospasm in all three sizes of arterial vessel size categories. The weight ratio to the contralateral muscle not subjected to I/R was greater in wild-type mice (173 +/- 11%) than in iNOS-/- mice (117 +/- 3%; P < 0.01). Inflammation and neutrophil extravasation were also more severe in wild-type mice. Western blot analysis demonstrated an absence of iNOS protein band in iNOS-/- mice and upregulation of iNOS protein expression in wild-type mice. Our results confirm the importance of iNOS in I/R injury. Upregulated iNOS exacerbates I/R injury and appears to be a therapeutic target in protection of tissues against this type of injury.     

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）在低氧性肺血管收缩中起着重要作用,低氧肺动脉高压下调内皮型。氧化氮合酶（endothelial nitric oxide synthase,eNOS）,使一氧化氮（nitric oxide,NO）的产量下降,但目前尚无关于15-HETE与eNOS／NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS／NO的相互作用进行研究。首先分离人鼠肺动脉,分为eNOS抑制剂L-NAME组（0．1mmol／L）、去缸管内皮组与内皮完整组,用15-HETE作用夫鼠离体肺动脉环,测定肺动脉张力。结果表明,L-NAME组、去除内皮组与内皮完整组分别比较,15-HETE对血管的收缩作用增强,且都有统计学意义（P〈0．05）。培养牛肺动脉内皮细胞,分别用15-HETE、15-脂氧酶（15-lipoxygenase,15-LO）抑制剂[（cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate,CDC）和（nordihydroguiairetic acid,YDGA）]处理细胞,通过Greiss方法检测亚硝酸盐含量,间接测定NO产量,与对照组比较,1μmol／L 15-HETE明显降低肺动脉内皮细胞NO水平（P〈0．05）,10μmol／L CDC和0．1mmol／L NDGA显著增加NO水平（分别是P〈0．05,P〈0．01）;通过Western blot检测不同时间（5,10,15,20,30,60min）eNOS的表达情况,结果显示,15-HETE的不同作用时间,没有引起eNOS表达的明显不同;用苏氨酸495位点磷酸化eNOS（Thr495）抗体进行免疫沉淀,再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量,问接测定eNOS活性,结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点,因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS／NO参与,15-HETE可以通过磷酸化Thr495位点降低eNOS活性,并且首次发现磷酸化eNOS（Thr495）和15-LO之间存在蛋白质相互作用。     

Regulation of prostaglandin generation in carrageenan-induced pleurisy by inducible nitric oxide synthase in knockout mice

In the present study, by comparing the responses in wild-type mice (iNOSWT) and mice lacking (iNOSKO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the correlation between endogenous nitric oxide (NO) and prostaglandin (PG) generation in carrageenan-induced pleurisy. The inflammatory response in iNOSKO mice was significantly reduced in respect to iNOSWT animals, as demonstrated by the exudate volume (-63%) and numbers of infiltrating cells (-62%). The levels of NOx in the pleural exudate from carrageenan-treated mice were significantly (p < 0.01) decreased in iNOSKO mice (16 +/- 7.6 nmoles/mice) compared to iNOSWT animals (133 +/- 9 nmoles/mice). Similarly, the amounts of PGE2 in the pleural exudates of carrageenan-treated animals were significantly (p < 0.01) lower in iNOSKO compared to iNOSWT mice (120 +/- 20 pg/mice vs. 308 +/- 51 pg/mice). Also the amounts of 6-keto-PGF(1 alpha) produced by lungs from carrageenan-treated iNOSKO mice (1.01 +/- 0.10 ng/tissue mg) were significantly (p < 0.01) reduced compared to iNOSWT carrageenan-treated mice (2.1 +/- 0.09 ng/tissue mg). In conclusion our results confirm, by the use of iNOSKO mice that in carrageenan-induced pleurisy NO positively modulates PG biosynthesis.