Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.

Diurnal variation of flow-mediated vasodilation in healthy premenopausal women.

The present study was designed to test the hypothesis of a diurnal variation of endothelial function. Sixteen healthy, nonsmoking women were studied, each on four occasions during one 24-h period (2:00 PM, 8:00 PM, 2:00 AM, and 8:00 AM). Endothelial function was assessed by ultrasound determinations of flow-mediated vasodilation (FMD%) in the brachial artery. FMD% was contrasted with endothelium-independent vasodilation, i.e., nitroglycerine-induced vasodilation (NTG%). Additionally, plasma concentrations and urinary excretion of nitrate and cGMP were analyzed. FMD% and NTG% displayed diurnal, albeit not parallel, patterns of variation. Whereas FMD% gradually increased from 2:00 PM and peaked at 2:00 AM (means +/- SE: 3.1 +/- 0.4, 4.4 +/- 0.4, 5.1 +/- 0.9, and 3.9 +/- 0.8%), the NTG% demonstrated a nadir at 2:00 AM. Plasma levels and urinary excretion of nitrate and cGMP did not display diurnal variation and no clear association with the variations seen in FMD% and NTG%. This study demonstrates a diurnal variation in both endothelium-dependent and -independent vasodilation in the brachial artery of healthy women. The background and possible implication of such a variation require further studies.

Correlation between increased nitric oxide production and markers of endothelial activation in systemic sclerosis: findings with the soluble adhesion molecules E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1.

OBJECTIVE: To determine the relationship between vascular function and the inflammatory response in systemic sclerosis (SSc), and to investigate whether production of endothelial-derived nitric oxide (NO) is disturbed in this disease. METHODS: We measured plasma nitrate, urinary excretion of both nitrate and cGMP, and soluble adhesion molecules of endothelial origin in patients with SSc and in age- and sex-matched controls and compared these levels between groups. Additionally, we performed correlation analysis to determine how these variables were related to one another. Plasma nitrate and 24-hour-urinary excretion of nitrate in patients and controls were measured after a 72-hour nitrate-free-diet, using a gas chromatography/mass spectrometric method. Soluble adhesion molecules intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1), and E-selectin and cytokines were measured by enzyme-linked immunosorbent assay. The expression of E-selectin was further investigated in skin biopsy specimens by immunoperoxidase staining, and the presence of inducible NO synthase by immunoblotting. RESULTS: Plasma nitrate and 24-hour-urinary-excretion of cGMP were significantly elevated in patients compared with controls, while 24-hour-urinary-excretion of nitrate tended to be elevated in SSc patients. Levels of sICAM-1, sVCAM-1, and sE-selectin were significantly elevated in the patients. Levels of plasma nitrate in the patients correlated significantly with levels of sVCAM-1 (P = 0.020) and sE-selectin (P = 0.018) and approached a significant correlation with sICAM-1 (P = 0.055), suggesting that activated endothelial cells may produce plasma nitrate. CONCLUSION: NO synthesis is elevated in SSc patients, and the activated endothelial cell is a likely site of its production.

Nitric oxide metabolites in CSF of patients with MS are related to clinical disease course.

In this study, we have analyzed the stable metabolites of nitric oxide (NO), nitrite and nitrate, in the CSF of patients with MS, lymphocytic meningitis, and in healthy control subjects. Patients with MS with an active disease course exhibited increased CSF nitrite levels compared with patients with stable MS and healthy control subjects, whereas CSF nitrate levels did not differ between these groups. High CSF levels of both metabolites were seen in patients with meningitis. These data indicate that CSF nitrite levels may reflect clinical disease activity in MS.

Detection of an approximately 100 kD protein with strong immunoreactivity to antibodies specific for inducible nitric oxide synthase (iNOS) but without NOS activity in neutrophils of patients suffering from sepsis: results of a preliminary study.

OBJECTIVE: The study was designed to evaluate the expression of inducible nitric oxide synthase (iNOS) in activated human neutrophils. SUBJECTS AND METHODS: By Western blotting and immunocytochemical staining, iNOS expression was analyzed in neutrophils from patients with sepsis who we classified by high plasma nitrate as subjects with activated NO metabolism. For comparison, rats treated with Zymosan documented to induce iNOS were analyzed. RESULTS: Strong immunoreactivity to antibodies for iNOS was detected in leukocytes of Zymosan treated rats and in neutrophils of septic patients. Such immunoreactivity was absent in untreated rats and healthy subjects. In rats, the immunoreactivity was associated to the 130 kD protein as expected for iNOS. In contrast, the intense iNOS staining in neutrophils of septic patients was associated to an approximately 100 kD protein. Despite the iNOS staining, no increased NOS activity could be demonstrated in the neutrophils of septic patients. CONCLUSIONS: The detection of an approximately 100 kD protein with immunoreactivity to antibodies recognizing human iNOS but without NOS activity in neutrophils of patients with sepsis should initiate studies to elucidate the origin and function of this protein.

Plasma nitrate as an index of nitric oxide formation in man: analyses of kinetics and confounding factors.

Nitric oxide (NO) is metabolized to nitrate in humans. Accordingly, plasma nitrate has been proposed as an index of the in vivo formation of NO. Such an application requires knowledge about the possible influence of nitrate from sources other than endogenous NO formation, as well as of the kinetics of nitrate in plasma. In the present study, plasma nitrate increased from 32 +/- 4 to 205 +/- 27 mumol/l (mean +/- SE) following intake of nitrate-rich food. It dropped during the intake of nitrate-restricted diet and stabilized at a level of 29 +/- 1 mumol/l. The urinary excretion of nitrate during nitrate restriction was 840 +/- 146 mumol/24 h. Plasma nitrate was not affected following the intake of a gastrointestinal antibiotic drug for a period of four days. Smoking three cigarettes in succession did not affect the plasma nitrate levels significantly. The oral intake of potassium nitrate (500 mg approximately 4950 mumol) elevated plasma nitrate from 29 +/- 3 to 313 +/- 12 mumol/l within 60 min. The subsequent drop in plasma nitrate, with a t1/2 of 451 +/- 42 min, was probably a reflection of the redistribution of nitrate within the body fluids and the renal excretion of nitrate. The plasma clearance of nitrate was 30 +/- 2 ml/min/1.73 m2 BSA. The distribution volume for nitrate was 28 +/- 1% of the bodyweight (BW). We conclude that plasma nitrate can be used as an index of the endogenous formation of NO, provided that the oral intake of nitrate is restricted for at least 48 h. Due to the large distribution volume and the low clearance of the ion wide-spread, marked, and chronic changes in NO formation are required to significantly affect the levels of nitrate in samples of mixed blood.

Nitric oxide-donating properties of mesoionic 3-aryl substituted oxatriazole-5-imine derivatives.

1. The nitric oxide (NO)-releasing properties of two new mesoionic 3-aryl substituted oxatriazole-5-imine derivatives (GEA 3162 and GEA 3175) were characterized and compared with the known NO-donors 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). 2. GEA 3162, GEA 3175, SIN-1 and SNAP inhibited adenosine 5'-diphosphate-induced platelet aggregation (IC50 values 0.18, 0.39, 3.73 and 2.12 microM, respectively). All four compounds induced a dose-dependent and more than 4 fold increase in cyclic GMP in platelets. The increase in cyclic GMP concentration was potentiated more than 1.5 fold by a phosphodiesterase inhibitor, zaprinast (10 microM) and inhibited 38-97% by oxyhaemoglobin (10-45 microM). 3. All of the four compounds studied converted oxyhaemoglobin to methaemoglobin and formed a paramagnetic NO-haemoglobin complex. All but GEA 3175 formed nitrite and nitrate in phosphate buffer. During a 40 min incubation, GEA 3162, SIN-1 and SNAP (100 microM) produced 50-70 microM NO2- + NO3- as determined by high performance liquid chromatography. The release of NO and NO2 by GEA 3175 was increased 140 fold in the presence of human plasma (0.14 and 19.7 ppb in the absence and presence of 1% human plasma, respectively) as analyzed by ozone chemiluminescence. 4. The results suggest that the mesoionic 3-aryl substituted oxatriazole-5-imine derivatives GEA 3162 and GEA 3175 as well as SIN-1 and SNAP release nitric oxide.

Cytokine-induced expression of nitric oxide synthase results in nitrosylation of heme and nonheme iron proteins in vascular smooth muscle cells.

Nitric oxide synthase (NOS) catalyzes the synthesis of the biomediator, nitric oxide (NO), from L-arginine. We have analyzed NOS induction and activity in cultured rat vascular smooth muscle cells (SMC), which respond to NO by relaxation and inhibition of mitochondrial respiration. Both interferon-gamma and tumor necrosis factor-alpha induced the expression of NOS mRNA and a combination of the two cytokines had a synergistic effect. An internal oligonucleotide complementary to murine macrophage NOS mRNA hybridized to polymerase chain reaction (PCR) products derived from SMC NOS but not brain NOS. Direct sequencing of the PCR products showed a high degree of homology between inducible NOS from SMC and macrophages. Analysis of NOS-dependent nitrite production demonstrated that the enzyme requires NADPH as a cofactor but not calcium for its activity. Cytokine treatment resulted in the development of electron paramagnetic resonance (EPR) signals characteristic for nitrosyl complexes, indicating nitrosylation of SMC molecules by enzymatically synthesized NO. De novo NOS gene transcription and protein synthesis are required for the cytokine-induced protein nitrosylation since addition of actinomycin D and cycloheximide abolished the cytokine effect. At an early stage of cytokine treatment and when low doses of cytokines were used, the EPR signal was dominated by a triplet hyperfine structure typical for hemenitrosyl complexes. With increasing incubation time and/or cytokine dose, the EPR spectra were gradually converted into a pattern resembling that of nonheme iron(II)-nitrosyl thiol complexes. Thereafter, the EPR signal shape no longer changed while the signal intensity increased quantitatively with NO synthesis, suggesting that considerable amounts of NO synthesized could be trapped in the cells by formation of nitrosyl complexes with intracellular molecules. Together, these results provide direct biochemical evidence for cytokine induction of NO synthesis and protein nitrosylation in SMC. This may represent an important second messenger system for cytokine effects on cellular metabolism in blood vessels.

Metabolism and excretion of nitric oxide in humans. An experimental and clinical study.

Despite the increasing insight in the clinical importance of nitric oxide (NO), formerly known as endothelium-derived relaxing factor (EDRF), there is limited information about the metabolism and elimination of this mediator in humans. We studied the degradation of NO in healthy subjects inhaling 25 ppm for 60 minutes and in patients with severe heart failure inhaling 20, 40, and 80 ppm in consecutive 10-minute periods. In other healthy subjects, the renal clearance of NO metabolite was measured. The metabolism ex vivo was evaluated by direct incubation of nitrite, the NO oxidation product, in blood from healthy humans. During inhalation of NO, the plasma levels of nitrate increased progressively, both in the healthy subjects (from 26 to 38 mumol/L, P < .001) and in the patients (from 72 to 90 mumol/L, P < .001). Methemoglobin (MetHb) also increased in the healthy subjects (from 7 to 13 mumol/L, P < .001) as well as in the patients (from 19 to 42 mumol/L, P < .01). No change in nitrosohemoglobin (HbNO) was detected, either in the healthy subjects or in the patients. In arterialized blood (O2 saturation, 94% to 99%), incubated nitrite was semiquantitatively converted to nitrate and MetHb. In venous blood (O2 saturation, 36% to 85%) moderate amounts of HbNO were also formed. Plasma and urinary clearance of nitrate in healthy subjects averaged 20 mL/min. We conclude that uptake into the red blood cells with subsequent conversion to nitrate and MetHb is a major metabolic pathway for endogenously formed NO. Nitrate may then enter the plasma to be eliminated via the kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Intranasal administration of human growth hormone (hGH) in combination with a membrane permeation enhancer in patients with GH deficiency: a pharmacokinetic study.

Recombinant human GH (hGH) combined with a permeation enhancer for the nasal mucosa, sodium tauro-24,25-dihydrofusidate (STDHF), was administered intranasally (in) in six patients with classical GH deficiency. Three different doses were tested (0.2, 0.4, and 0.8 IU/kg BW). The concentration of STDHF was 1% in all doses. As a comparison, all patients received a sc injection of hGH (0.1 IU/kg BW). Blood samples were obtained at frequent intervals for up to 8 h (in doses) or 24 h (sc dose) and analyzed for the plasma concentration of hGH. All three i.n. doses gave a rapid increase in hGH with peak maxima (Cmax) at 20-30 min, and a decline to baseline within 2-3 h. In contrast, the sc dose resulted in a Cmax 2-3 h after the injection, followed by a plateau phase for 2-3 h. The baseline was reached 12-14 h after administration. The uptake [area under the curve (AUC)] was considerably lower for all three in doses, i.e. 1.6-3% of the AUC obtained with the sc dose. However, the Cmax varied between 5.7 +/- 1.4% (0.8 IU/kg BW) and 15.8 +/- 2.1% (0.4 IU/kg BW) of the maximal peak with the sc dose. Of the in doses, 0.4 IU/kg BW resulted in the highest AUC and Cmax. A self-rating protocol was also used to estimate nasal sensations for up to 2 h after dosing. All sensations (itching, burning, sneezing, and running of the nose) were transient and tolerable. This study demonstrates that hGH can be administered intranasally in combination with STDHF. The in dosing results in a plasma peak of hGH very similar to the physiological endogenous peak. No side-effects were noted, other than a transient nasal irritation. Therefore, the nasal route for hGH administration offers a more physiological and more convenient alternative to injections for the treatment of GH deficiency.

Plasma nitrate as an index of immune system activation in animals and man.

The cytotoxic activity of activated macrophages is based upon their formation and release of nitric oxide (NO). In blood NO is rapidly metabolised to nitrate. We analysed plasma nitrate in rats given zymosan, an activator of the immune system, and found 20-fold increases above the basal level. Furthermore, in two human subjects with acute gastro-enteritis plasma nitrate increased from about 30 to 200 microM two days after onset of symptoms. These increased plasma levels of nitrate may reflect macrophage formation of NO. We propose that plasma nitrate may be a useful index for quantitative assessment of cytotoxic activity during immunological challenge.

Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin.

Plasma or whole venous or arterialized blood from healthy human donors was incubated with NO (50-300 microM), and the resulting formation of methaemoglobin (MetHb), nitrosyl haemoglobin (HbNO), and plasma nitrite and nitrate were measured. In plasma, NO was converted to nitrite and nitrate in a ratio of 5:1. In arterial blood (O2 sat. 94-99%) NO was almost quantitatively converted to nitrate and MetHb. No nitrite was detected and HbNO formation was low. In venous blood (O2 sat. 36-85%) more HbNO and less nitrate was formed, in comparison to arterialized blood. We propose that NO liberated from endothelium of conductance and resistance vessels is taken up by red blood cells and inactivated by HbO2 via stoichiometric conversion to MetHb and nitrate.

Relation between tobacco use and urinary excretion of thromboxane A2 and prostacyclin metabolites in young men.

BACKGROUND: Cigarette smoking is a risk factor for cardiovascular disease. The present study addressed the effect of tobacco use on the formation of two eicosanoids, thromboxane A2 and prostacyclin, which have been implicated in both acute and chronic cardiovascular disorders. METHODS AND RESULTS: In 577 randomly sampled 18-19-year-old men, the urinary excretion of the 2,3-dinor metabolites of thromboxane A2 and prostacyclin (Tx-M and PGI-M, respectively) was analyzed and related to the subjects' self-reported use of tobacco. Sixty-five percent of the subjects used no tobacco, 7.5% were cigarette smokers, 22% used wet (oral) snuff, and the rest reported a mixed use of tobacco. The urinary excretion of Tx-M was higher (p less than 0.001) in cigarette smokers than in those not using tobacco (180 versus 128 pg/mg creatinine) and was correlated (r = 0.35, p less than 0.05) with the daily cigarette consumption. Snuff users had no increase in their urinary excretion of Tx-M, despite urinary cotinine levels comparable to those in the cigarette smokers (1,210 and 1,560 ng/ml, respectively). The excretion of PGI-M did not differ between non-tobacco users, cigarette smokers, and snuff users. CONCLUSIONS: We conclude that cigarette smoking, but not the use of snuff, facilitates the formation of thromboxane A2. We propose that such an increased formation reflects platelet activation in the absence of vascular injury and that it may be of significance for the subsequent development of cardiovascular disease.

Release of endothelial mediators and sympathetic transmitters at different coronary flow rates in rabbit hearts.

1. The release of three endothelial mediators, namely, endothelial-derived relaxing factor (EDRF), prostacyclin (PGI2) and endothelin, and of two sympathetic neurotransmitters, noradrenaline and neuropeptide Y (NPY), from resting or sympathetically stimulated rabbit Langendorff hearts was investigated at normal or elevated coronary flow. The sympathetic nerves to the hearts were stimulated at 5 Hz for 30 s and the cardiac effluent was analysed for nitrite (metabolite of EDRF) with electron paramagnetic resonance spectrometry, for 6-keto-PGF1 alpha (metabolite of PGI2) with gas chromatography/mass spectrometry, for endothelin- and NPY-like immunoreactivity with radioimmunoassay, and for noradrenaline and purines with liquid chromatography. 2. During perfusion of the hearts at normal flow (35 +/- 1.4 ml min-1) the effluent concentration of nitrite was 0.15 +/- 0.02 microM, that of 6-keto-PGF1 alpha 0.74 +/- 0.08 nM, and that of endothelin-like immunoreactivity 0.18 +/- 0.01 pM. Nerve stimulation augmented the release of 6-keto-PGF1 alpha from 76 +/- 8 to 99 +/- 10 pmol (3 min)-1 (P less than 0.05), but did not affect the release of nitrite or endothelin-like immunoreactivity. Nerve stimulation also facilitated the outflow of noradrenaline and of NPY-like immunoreactivity by 52 +/- 11 pmol (3 min)-1 and 19 +/- 7 fmol (3 min)-1, respectively. 3. Elevation of the coronary flow to 79 +/- 3.2 ml min-1 did not affect the effluent concentrations of nitrite, 6-keto-PGF1 alpha and endothelin-like immunoreactivity, implying that their outflows were augmented. Sympathetic stimulation at elevated coronary flow did not further augment the outflow of endothelial mediators or of NPY-like immunoreactivity, but increased the outflow of noradrenaline by 62 +/- 12%, in comparison to stimulation at normal flow. Perfusion of the heart with the noradrenaline uptake blocker desipramine (5 microM) completely abolished the promoting effecting of elevated coronary flow on noradrenaline outflow during sympathetic stimulation. 4. These data indicate that an increase in coronary flow in perfused rabbit hearts is paralleled by a corresponding facilitation of the formation of the endothelial mediators, EDRF, prostacyclin and endothelin. Such an elevation of mediator formation does not affect nerve stimulation-induced release of sympathetic transmitters in the heart.