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.     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.     

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.     

Involvement of inducible nitric oxide synthase in cardiac dysfunction with tumor necrosis factor-alpha

Transgenic (TG) mice with cardiac-specific overexpression of tumor necrosis factor (TNF)-alpha develop dilated cardiomyopathy with myocardial inflammation. The purpose of this study was to investigate the role of nitric oxide (NO) in this mouse model of cardiomyopathy. Female TG and wild-type mice at the age of 10 wk were studied. The expression and activity of inducible NO synthase (iNOS) were significantly increased in the TG myocardium, whereas those of endothelial NOS were not altered. The majority of the iNOS protein was isolated in the interstitial cells. The selective iNOS inhibitor (1S,5S,6R,7R)- 7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714) was used to examine the effects of iNOS induction on myocardial contractility. Echocardiography and left ventricular pressure measurements were performed. Both fractional shortening and the maximum rate of rise of left ventricular pressure were significantly suppressed in TG mice. Although ONO-1714 did not change hemodynamic parameters or contractility at baseline, it significantly improved beta-adrenergic inotropic responsiveness in TG mice. These results indicate that induction of iNOS may play an important role in the pathogenesis of cardiac dysfunction in this mouse model of cytokine-induced cardiomyopathy.     

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.     

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle.

Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast-twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscle's contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi-innervated fibers. The NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S-nitroso-N-acetylcysteine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOM's specialized role in generation of eye movements.     

Levels of protein kinase C and nitric oxide synthase activity in rats exposed to sub chronic low level lead

Previous studies from our laboratories have linked the protective abilities of IH636 grape seed proanthocyanidin extract (GSPE) with inactivation of anti-apoptotic gene bcl-XL, and modification of several other critical molecular targets such as DNA-damage/DNA-repair, lipid peroxidation and intracellular Ca²⁺ homeostasis. Especially, GSPE provided dramatic protection against acetaminophen (APAP)-induced hepatotoxicity, significantly increased bcl-XL expression in the liver [1], and antagonized both necrotic and apoptotic deaths of liver cells in vivo. However, it was not clear from this study whether anti-apoptogenic and anti-necrotic effects of GSPE were: (i) due to its interference with endonuclease activity, (ii) due to its antioxidant effect, or, (iii) due to its ability to inhibit microsomal drug metabolizing enzyme(s), such as CYP-4502E1. Since CYP-4502E1 primarily metabolizes acetaminophen in mice and rats, this study specifically focused on CYP-4502E1's catalytic activity in vitro. Overall this investigation compared the in vitro aniline hydroxylation patterns of: (i) in vivo GSPE-exposed and unexposed (control) mouse liver microsomes, (ii) induced (1% acetone in drinking water for 3 days) and uninduced rat liver microsomes in the presence and absence of GSPE in vitro, and (iii) control rat liver microsomes in the presence of an anti-APAP agent 4-aminobenzamide (4-AB) in vitro. For the in vivo assessment, male B6C3F1 mice were fed GSPE diet (ADI 100 mg/kg body wt) for 4 weeks, and liver microsomes were isolated from both control and GSPE-fed mice for aniline hydroxylation, a specific marker of CYP-4502E1 activity. Data show that hydroxylation was 40% less in microsomes from GSPE-exposed livers compared to control microsomes. Similarly, when rat liver microsomes were incubated with various concentrations of GSPE in vitro (100 and 250 g/ml), aniline hydroxylation was inhibited to various degrees (uninduced: 40 and 60% and induced: 25 and 50%, respectively with 100 and 250 g/ml). Influence of GSPE on hydroxylation patterns were compared with another hepatoprotective agent 4-aminobenzamide (4-AB), a well-known modulator of nuclear enzyme poly(ADP-ribose) polymerase, and the data shows that 4-AB did not alter aniline hydroxylation at all. Collectively, these results may suggest that GSPE has the ability to inhibit CYP-4502E1, and this is an additional cytoprotective attribute, in conjunction with its novel antioxidant and/or antiendonucleolytic potential.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Involvement of endogenous nitric oxide in angiotensin II-induced activation of vascular mitogen-activated protein kinases

Angiotensin II (ANG II) is a powerful activator of mitogen-activated protein (MAP) kinase cascades in cardiovascular tissues through a redox-sensitive mechanism. Nitric oxide (NO) is considered to antagonize the vasoconstrictive and proarteriosclerotic actions of ANG II. However, the role of endogenous NO in ANG II-induced redox-sensitive signal transduction is not yet clear. In this study using catheterized, conscious rats, we found that acute intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg) enhanced phosphorylation of aortic MAP kinases extracellular signal regulated kinase (ERK) 1/2 and p38, which were suppressed only partially by a superoxide dismutase mimetic (Tempol), whereas ANG II-induced MAP kinase phosphorylation was markedly suppressed by Tempol. FK409, a NO donor, had little effect on vascular MAP kinase phosphorylation. On the other hand, acute exposure to a vasoconstrictor dose of ANG II (200 ng x kg(-1) x min(-1) iv) failed to enhance phosphorylation of aortic MAP kinases in the chronically L-NAME-treated rats, whereas the vasoconstrictor effect of ANG II was not affected by L-NAME treatment. Furthermore, three different inhibitors of NO synthase suppressed, in a dose-dependent manner, ANG II-induced MAP kinase phosphorylation in rat vascular smooth muscle cells, which was closely linked to superoxide generation in cells. These results indicate the involvement of endogenous NO synthase in ANG II-induced signaling pathways, leading to activation of MAP kinase, and that NO may have dual effects on the vascular MAP kinase activation associated with redox sensitivity.     

Antioxidant and nitric oxide synthase activation properties of Auricularia auricula

In vitro evaluation of antioxidant activities of Auricularia auricula showed significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with standard drug catechin. IC5o value of crude, boiled and ethanolic extracts of A. auricula represented 403, 510, and 373 microg/ml respectively in case of hydroxyl radical scavenging activity and 310, 572 and 398 microg/ml respectively in case of lipid peroxidation. Furthermore, crude, boiled and ethanolic extracts also increase significantly nitric oxide production (664, 191 and 850 pmole/mg dry wt/hr respectively) over the control. The present results revealed that A. auricula had potential therapeutic use.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Angiotensin II-induced decrease in expression of inducible nitric oxide synthase in rat astroglial cultures: role of protein kinase C

Inducible nitric oxide synthase (iNOS) has been implicated as a mediator of cellular toxicity in a variety of neurodegenerative disorders. Nitric oxide, which is generated in high quantities following induction of iNOS, combines with other oxygen radicals to form highly reactive, death-inducing compounds. Given the frequency of neuronal death due to neurodegenerative diseases, cerebral trauma, and stroke, it is important to study the mechanisms of regulation of iNOS in the brain. We demonstrated previously that angiotensin II (Ang II) decreases the expression of iNOS produced by bacterial endotoxin or cytokines in cultured astroglia prepared from adult rat brain. Here, we have addressed the mechanisms by which Ang II negatively modulates iNOS. The inhibitory effects of Ang II on lipopolysaccharide-induced expression of iNOS mRNA and protein and nitrite accumulation were mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate. Down-regulation of PKC produced by long-term treatment of astroglia with phorbol 12-myristate 13-acetate abolished the inhibitory effect of Ang II on lipopolysaccharide-stimulated expression of iNOS mRNA and nitrite accumulation. Finally, the reduction of lipopolysaccharide-induced nitrite accumulation by Ang II was attenuated by the selective PKC inhibitor chelerythrine. Collectively, these data indicate a role for PKC in the inhibitory actions of Ang II on iNOS expression in cultured astroglia.     

Attenuation of mouse mesangial cell contractility by high glucose and mannitol: Involvement of protein kinase C and focal adhesion kinase

Hyperglycemia and mannitol activate protein kinase C (PKC) and induce mesangial cell hypocontractility that subsequently may modulate renal function. Since focal adhesion kinase (FAK) activation is known to be linked with PKC activity, FAK may also be involved in mesangial cell contraction. To facilitate our understanding of the PKC- and FAK-modulating mechanism, we developed an in vitro model of mouse mesangial cell hypocontractility induced by hyperglycemia or mannitol. Mouse mesangial cells (CRL-1927) were exposed to: normal D-glucose (group N), high D-glucose (group H), and control groups at the same osmolality as H plus L-glucose (group L) and mannitol (group M). Changes in the planar surface area of cells in response to 1 microM phorbol 12-myristate 13-acetate (PMA) were determined. Western blot analyses for PKC, phosphorylated (p)-PKC, tyrosine phosphorylation, FAK, and p-FAK were done on each of these four groups. The effects of mannitol in various doses on cell contraction and activation of PKC and FAK were also assayed. The planar surface areas of groups H and M both showed an attenuated change in response to PMA stimulation. Before PMA stimulation, the baseline PKC expression of groups H and M showed a higher expression of p-PKC alpha and delta than that seen in group N (p < 0.05). Results of tyrosine phosphorylation and immunoprecipitation showed that FAK may be involved in this contraction process. The total amount of FAK showed no significant difference among the four experimental groups; however, p-FAK was found to have significantly increased in group M (p < 0.05). The use of PKC and tyrosine kinase inhibitors reduced PMA-induced mesangial cell contraction in all four groups. Activation of PKC alpha, delta, and FAK with the resultant inhibition of mesangial cell contraction by mannitol was found to be dose-dependent. These results may provide a correlation between increased expression of several PKC isoforms and, in particular, increased phosphorylation levels of PKC alpha and delta and hypocontractility induced by high glucose and mannitol treatment. Furthermore, the mannitol-induced hypocontractility involving PKC and FAK occurred in a dose-dependent manner.     

Differential expression of protein kinase C isoenzymes related to high nitric oxide synthase activity in a T lymphoma cell line

Protein kinase C (PKC) is critical for T lymphocyte activation and proliferation, while nitric oxide synthase (NOS) may function both as an activator or inhibitor of T cell apoptosis. Both enzymatic activities were studied in T lymphoma cells in comparison to normal and activated T lymphocytes. Here we show a higher translocation of PKC in BW5147 lymphoma cells than in mitogen-stimulated T lymphocytes. Tumor cells overexpressed PKC zeta isoform, while high levels of the PKC beta isotype were found in mitogen-stimulated T lymphocytes. Moreover, tumoral T cells showed high NOS activity, almost undetectable in normal or stimulated T lymphocytes. PKC and NOS inhibitors or the intracellular delivery of an anti-PKC zeta antibody diminished both NO production and proliferation in tumor cells.These results suggest that atypical PKC zeta isoform expression and its association with NOS activity regulation would participate in the multistep process leading to BW5147 cell malignant transformation.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.     

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.