L-arginine chlorination products inhibit endothelial nitric oxide production

The myeloperoxidase-derived oxidant hypochlorous acid (HOCl) is thought to contribute to endothelial dysfunction, but the mechanisms underlying this inhibitory effect are unknown. The present study tested the hypothesis that HOCl and L-arginine (L-Arg) react to form novel compounds that adversely affect endothelial function by inhibiting nitric oxide (NO) formation. Using spectrophotometric techniques, we found that HOCl and L-Arg react rapidly (k = 7.1 x 10(5) m(-1) s(-1)) to form two major products that were identified by mass spectrometry as monochlorinated and dichlorinated adducts of L-Arg. Pretreatment of bovine aortic endothelial cells with the chlorinated L-Arg metabolites (Cl-l-Arg) inhibited the -induced formation of the NO metabolites nitrate (NO(3)(-)) and nitrite (NO(2)(-)) in a concentration-dependent manner. Preincubation of rat aortic ring segments with Cl-L-Arg resulted in concentration-dependent inhibition of acetylcholine-induced relaxation. In contrast, blood vessels relaxed normally to the endothelium-independent vasodilator sodium nitroprusside. In vivo administration of Cl-L-Arg to anesthetized rats increased carotid artery vascular resistance. A greater than 10-fold excess of L-Arg was required to reverse the inhibitory effects of Cl-L-Arg in vivo and in vitro. Reaction of HOCl with D-arginine (D-Arg) did not result in the formation of inhibitory products. These results suggest that HOCl reacts with L-Arg to form chlorinated products that act as nitric-oxide synthase inhibitors.     

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.     

Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 10(5)) was about 20 times greater than that of YkuP (2.6 x 10(4)), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs.     

High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells

Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.     

Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.     

Phosphorylation of tyrosine 801 of vascular endothelial growth factor receptor-2 is necessary for Akt-dependent endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells

Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-gamma in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGF-stimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.     

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.     

Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase

We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.     

Coordinate regulation of L-arginine uptake and nitric oxide synthase activity in cultured endothelial cells.

Despite intracellular L-arginine concentrations that should saturate endothelial nitric oxide synthase (eNOS), nitric oxide production depends on extracellular L-arginine. We addressed this 'arginine paradox' in bovine aortic endothelial cells by simultaneously comparing the substrate dependence of L-arginine uptake and intracellular eNOS activity, the latter measured as L-[3H]arginine conversion to L-[3H]citrulline. Whereas the Km of eNOS for L-arginine was 2 microM in cell extracts, the L-arginine concentration of half-maximal eNOS stimulation was increased to 29 microM in intact cells. This increase likely reflects limitation by L-arginine uptake, which had a Km of 108 microM. The effects of inhibitors of endothelial nitric oxide synthesis also suggested that extracellular L-arginine availability limits intracellular eNOS activity. Treatment of intact cells with the calcium ionophore A23187 reduced the L-arginine concentration of half-maximal eNOS activity, which is consistent with a measured increase in L-arginine uptake. Increases in eNOS activity induced by several agents were closely correlated with enhanced L-arginine uptake into cells (r = 0.89). The 'arginine paradox' may be explained in part by regulated L-arginine uptake into a compartment, probably represented by caveolae, that contains eNOS and that is distinct from the bulk cytosolic L-arginine.     

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.     

Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration. The protein kinase Akt activates the endothelial NO synthase (eNOS) by phosphorylation of Ser-1177. Therefore, we investigated the contribution of Akt-mediated eNOS phosphorylation to VEGF-induced EC migration. Inhibition of NO synthase or overexpression of a dominant negative Akt abrogated VEGF-induced cell migration. In contrast, overexpression of constitutively active Akt was sufficient to induce cell migration. Moreover, transfection of an Akt site phospho-mimetic eNOS (S1177D) potently stimulated EC migration, whereas a non-phosphorylatable mutant (S1177A) inhibited VEGF-induced EC migration. Our data indicate that eNOS activation via phosphorylation of Ser-1177 by Akt is necessary and sufficient for VEGF-mediated EC migration.     

Regulation of the monomer-dimer equilibrium in inducible nitric-oxide synthase by nitric oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.     

Apelin activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas

Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the L-arginine (L-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO(2)(-) product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10(-9), 10(-8), and 10(-7)mol/L) increased NO(2)(-) product by 33%, 46%, and 69% (all p<0.01), respectively, and Ca(2+)-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p<0.01), respectively. However, Ca(2+)-independent NOS (iNOS) activity was not significantly altered (p>0.05). Apelin incubation (10(-9), 10(-8), and 10(-7)mol/L) increased L-Arg uptake by 130%, 180%, and 240% (all p<0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10(-7)mol/L apelin was increased by 110% and 128%, respectively (both p<0.01). Incubation with 10(-7)mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p<0.05) and 319% (p<0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular L-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.     

Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase

The balance of nitric oxide (.NO) and superoxide anion (O(2)) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates.NO. As eNOS is capable of generating both.NO and O(2), we hypothesized that Hsp90 might also mediate eNOS-dependent O(2) production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 microg/ml; 17.8 microm) and then stimulated with the calcium ionophore, (5 microm). GA significantly decreased -stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased -stimulated eNOS-dependent O(2) production (p < 0.001, n = 8). increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent.NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O(2) production.     

Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase

Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2) intermediate, indicating that BH(4) indeed supplies the second electron for L-arginine monooxygenation in eNOS.     

Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in Escherichia coliand purified. The conversion of L-arginine to N(omega)-hydroxy-L-arginine and further to L-citrulline in one cycle of the reaction of the purified nNOS was measured with the reaction rapid quenching method using (3)H-L-arginine as the substrate. It was found that most of the produced (3)H-N(omega)-hydroxy-L-arginine was successively hydroxylated to (3)H-L-citrulline without leaving the enzyme. From the analysis of time courses, the rate constants for each reaction step, and also for the dissociation of the intermediate, were estimated at various temperature in which the rates for the first and the second reactions were not much different each other but the rate for the dissociation of (3)H-N(omega)-hydroxy-L-arginine from the enzyme was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst formation of L-citrulline in the pre-steady state. The rate constant for the dissociation of the product L-citrulline from nNOS was calculated from the combination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. The activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate-determining step of the nNOS reaction in the steady state was the dissociation of the product L-citrulline from the enzyme.     

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.     

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.     

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.     

Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox

L-Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K(M) values of these enzymes. Therefore, additional L-arginine should not enhance NO formation. In vivo, however, increasing L-arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L-arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC(50) 1.5 microM), are responsible for the L-arginine paradox is presented.