Protein kinase Cdelta regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

In this study, we explore the roles of the delta isoform of PKC (PKCdelta) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCdelta with either rottlerin or with the peptide, deltaV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCdelta inhibition using either rottlerin or the overexpression of a dominant negative PKCdelta mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCdelta inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCdelta is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCdelta-mediated Akt activation and NO generation in maintaining eNOS expression.     

Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning

The opening of mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels has a significant role in delayed ischemic preconditioning, and nitric oxide (NO) is a well-known trigger for its activation. However, the source of NO remains unknown. Phosphorylation of endothelial NO synthase (eNOS) increases NO production and reduces apoptosis through the Akt signaling pathway. To elucidate the Akt signaling pathway involved in the opening and antiapoptotic effect of mitoKATP channel during delayed pharmacological preconditioning, the mitoKATP channel opener diazoxide (DE, 7 microg/kg i.p.) alone or DE plus Nomega-nitro-L-arginine methyl ester (L-NAME, 30 microg/kg i.v.), an inhibitor of NOS, or wortmannin (WTN, 15 microg/kg i.v.), an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), was administered to wild-type (WT) or eNOS(-/-) mice during DE treatment. Twenty-four hours later, hearts were isolated and subjected to 40 min ischemia and 30 min reperfusion (I/R). The effect of DE and other interventions on hemodynamic, terminal dUTP nick-end labeling staining and biochemical changes during I/R was assessed in mouse hearts. Treatment with DE resulted in a 2.2-fold increase in phosphorylation of Akt and a significant increase in eNOS and inducible NOS (iNOS) proteins. Akt is upstream of NOS and the mitoKATP channel as simultaneous pretreatment of WTN with DE abolished phosphorylation of Akt, which was not affected by L-NAME and 5-hydroxydecanoate. In hearts treated with DE, cardiac function was significantly improved after I/R, and apoptosis was also significantly decreased. WTN abolished the antiapoptotic effect of DE. Similarly, S-methylisothiourea, a specific iNOS inhibitor, when given to eNOS(-/-) mice that were pretreated with DE completely abolished the beneficial effects of DE on reduction of apoptotic death. DE was partially effective in eNOS(-/-) mice against the ischemic injury. It is concluded that DE activates Akt through the PI3 kinase signaling pathway and iNOS and eNOS is downstream of Akt.     

Regulation of endothelial nitric oxide synthase by protein kinase C

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.     

AMP-activated protein kinase mediates erythropoietin-induced activation of endothelial nitric oxide synthase

We investigated whether AMP-activated protein kinase (AMPK), a multi-functional regulator of energy homeostasis, participates in the regulation of erythropoietin (EPO)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with EPO increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the EPO-induced increase in the phosphorylation of AMPK, ACC, and eNOS, as well as nitric oxide (NO) production. Additionally, suppression of AMPK activation abolished EPO-induced EC proliferation, migration and tube formation. Immunoprecipitation analysis demonstrated that AMPK mediated the EPO-induced increase in the phosphorylation of β common receptor (βCR) and the formation of a βCR-AMPK-eNOS complex. In mice, inhibition of AMPK activation by compound C markedly decreased EPO-elicited angiogenesis in Matrigel plugs. Furthermore, the phosphorylation of AMPK and eNOS was significantly higher in aortas from EPO transgenic mice than wild-type mice. Moreover, treatment with EPO neutralizing antibody greatly reduced the exercise training-induced increase in phosphorylation of AMPK and eNOS in aortas of wild-type mice. Taken together, EPO may trigger AMPK-dependent signaling, which leads to enhanced phosphorylation of βCR and eNOS, increased βCR-AMPK-eNOS complex formation, NO production, and, ultimately, angiogenesis.     

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

c-Jun N-terminal kinase 2 phosphorylates endothelial nitric oxide synthase at serine 116 and regulates nitric oxide production

The c-Jun N-terminal kinases (JNKs) belonging to the mitogen-activated protein kinase (MAPK) superfamily play important roles in foam-cell formation, hypercholesterolemia-mediated endothelial dysfunction, and the development of obesity. Although decreased nitric oxide (NO) production via decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser(1179)) was reported to be partly involved in JNK2-derived endothelial dysfunction, JNK2 seems likely to be indirectly involved in this signaling pathway. Here, using bovine aortic endothelial cells, we examined whether JNK2 directly phosphorylated eNOS-Ser(116), a putative substrate site for the MAPK superfamily, and this phosphorylation resulted in decreased NO release. JNK inhibitor SP60012 increased NO release in a time- and dose-dependent manner, which was accompanied by increased eNOS-Ser(116) phosphorylation. Purified JNK2 directly phosphorylated eNOS-Ser(116)in vitro. Ectopic expression of dominant negative JNK2 repressed eNOS-Ser(116) phosphorylation and increased NO production. Coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and JNK2. However, all these observed effects were not manifested when JNK1 probes were used. Overall, this study indicates that JNK2 is a physiological kinase responsible for eNOS-Ser(116) phosphorylation and regulates NO production.     

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.     

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.     

Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes

Nitric oxide (NO) is a major regulator of the cardiovascular system. However, the effects of endothelial nitric oxide synthase (eNOS) gene polymorphisms or haplotypes on the circulating concentrations of nitrite (a sensitive marker of NO formation) and cGMP are unknown. Here we examined the effects of eNOS polymorphisms in the promoter region (T-786C), in exon 7 (Glu298Asp), and in intron 4 (4b/4a) and eNOS haplotypes on the plasma levels of nitrite and cGMP. We hypothesized that eNOS haplotypes could have a major impact on NO formation. We genotyped 142 healthy subjects by PCR-RFLP. To assess NO formation, the plasma concentrations of nitrite and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Haplotypes were inferred using the PHASE 2.1 program. No significant differences were found in age, body mass index, systolic and diastolic arterial blood pressure, heart rate, total cholesterol, triglycerides, cGMP, or nitrite among the genotype groups for the three polymorphisms studied here (all p>0.05). Interestingly, the C-4b-Glu haplotype was associated with lower plasma nitrite concentrations than those found in the other haplotype groups (p<0.05), but not with different cGMP levels (p>0.05). These findings suggest that eNOS gene variants combined within a specific haplotype modulate NO formation, although individual eNOS polymorphisms probably do not have major effects.     

Rapid activation of endothelial nitric oxide synthase by estrogen.

Estrogen is an important atheroprotective molecule that causes the rapid dilation of blood vessels by stimulating endothelial nitric oxide synthase (eNOS). There is also evidence that estrogen modulates airway epithelial NO production, thereby potentially affecting bronchial hyperresponsiveness. Studies in cultured endothelial and airway epithelial cells indicate that physiologic concentrations of estrogen cause rapid direct activation of eNOS that is unaffected by actinomycin D, but fully inhibited by estrogen receptor (ER) antagonism. Overexpression of ERalpha leads to marked enhancement of the acute response to estrogen, and this process is blocked by ER antagonism, it is specific to estrogen, and it requires the ERalpha hormone binding domain. In addition, the acute response of eNOS to estrogen can be reconstituted in COS-7 cells cotransfected with wild-type ERalpha and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of calcium influx, or tyrosine kinases or MAP kinase prevents the stimulation of eNOS by estrogen, and estrogen causes rapid ER-dependent activation of MAP kinase. These findings indicate that the acute effects of estrogen on both endothelial and airway epithelial eNOS are mediated by ERalpha functioning in a novel, nongenomic manner to activate the enzyme via calcium-dependent, MAP kinase-dependent mechanisms.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Phosphorylation of nitric oxide synthase by protein kinase A.

Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.     

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.     

Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications.

The nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and NO through consumption of oxygen bound to the heme. Because NO is produced close to the heme and may bind to it, its subsequent role in a regulatory mechanism should be scrutinized. We therefore examined the kinetics of NO rebinding after photodissociation in the heme pocket of human endothelial NOS by means of time-resolved absorption spectroscopy. We show that geminate recombination of NO indeed occurs and that this process is strongly modulated by L-Arg. This NO rebinding occurs in a multiphasic fashion and spans over 3 orders of magnitude. In both ferric and ferrous states of the heme, a fast nonexponential picosecond geminate rebinding first takes place followed by a slower nanosecond phase. The rates of both phases decreased, whereas their relative amplitudes are changed by the presence of L-Arg; the overall effect is a slow down of NO rebinding. For the isolated oxygenase domain, the picosecond rate is unchanged, but the relative amplitude of the nanosecond binding decreased. We assigned the nanosecond kinetic component to the rebinding of NO that is still located in the protein core but not in the heme pocket. The implications for a mechanism of regulation involving NO binding are discussed.     

血管内皮型一氧化氮合酶的调节机制

血管内皮型一氧化氮合酶(eNOS)的调控机制可分为基因表达水平调节和蛋白水平调节两个方面。其中,eNOS的基因表达水平调节主要包含启动子的调节和mRNA的稳定性调节两方面。而eNOS的蛋白水平调节又可分为三个方面:eNOS细胞内转位的调节机制;eNOS复合体形成的调节机制;eNOS氨基酸残基磷酸化的调节机制。eNOS的分子调控机制与临床疾病的发生、发展及其治疗有着密切的关系,故对eNOS分子调控机制的进一步了解有着非常重要的意义。     

Modulation of endothelial nitric oxide synthase by fibronectin

Nitric oxide (NO) produced by the action of endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone, cell survival, and angiogenesis. Interaction of endothelial cells (ECs) with a fibronectin (FN) rich matrix is important in the regulation of EC function and survival during angiogenesis. The present study was carried out to examine if FN can regulate eNOS and thereby NO levels in ECs. The activity and the levels of mRNA and protein of eNOS were significantly low in HUVECs maintained in culture on FN. Inhibition of p38 MAPK and blocking the interaction of FN with α₅β₁ integrin using antibody caused the reversal of the FN effect. Immunoblot analysis of Ser/Thr phosphorylation of purified eNOS suggested that FN downregulates post-translational phosphorylation of eNOS at Ser residues. These results suggest that FN negatively modulates eNOS in an α₅β₁ integrin-p38 MAPK-dependent pathway.     

Downregulation of human endothelial nitric oxide synthase promoter activity by p38 mitogen-activated protein kinase activation.

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. eNOS gene expression may be upregulated by a signaling pathway, including PI-3Kgamma--> Jak2--> MEK1 --> ERK1/2--> PP2A. It remains unclear whether other mitogen-activated protein kinase (MAPK) family members, such as JNK, p38 kinase, and ERK5/BMK1, also modulate eNOS gene expression. Our purpose, therefore, is to shed light on the effect of the p38 MAPK signaling pathway on the regulation of eNOS promoter activity. The results showed that a red fluorescent protein reporter gene vector containing the full length of the human eNOS promoter was first successfully constructed, expressing efficiently in ECV304 cells with the characteristics of real time observation. The wild-types of p38alpha, p38beta, p38gamma, and p38delta signal molecules all markedly downregulated promoter activity, which could be reversed by their negative mutants, including p38alpha (AF), p38beta (AF), p38gamma (AF), and p38delta (AF). Promoter activity was also significantly downregulated by MKK6b (E), an active mutant of an upstream kinase of p38 MAPK. The reduction in promoter activity by p38 MAPK could be blocked by treatment with a p38 MAPK specific inhibitor, SB203580. Moreover, the activation of endogenous p38 MAPK induced by lipopolysaccharide resulted in a prominent reduction in promoter activity. These findings strongly suggest that the activation of the p38 MAPK signaling pathway may be implicated in the downregulation of human eNOS promoter activity.