Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia

OBJECTIVE: Nitric oxide is a potent vasorelaxant produced by endothelial cells. We tested the hypothesis that urinary and perhaps plasma nitric oxide metabolites would be reduced in women with preeclampsia. STUDY DESIGN: Plasma and urine from 14 women meeting strict clinical criteria for the diagnosis of preeclampsia and 20 normal nulliparous women were assayed for the stable metabolites of nitric oxide, nitrate and nitrite. RESULT: There was no significant difference of plasma concentrations of nitrate and nitrite between women with preeclampsia and women with normal pregnancies (32.7 +/- 3.1 vs 25.8 +/- 2.4 micromol/L). Plasma creatinine levels were elevated in women with preeclampsia (0.85 +/- 0.09 vs 0.66 +/- 0.02 mg/dl, p<0.01), indicating a reduced glomerular filtration rate. Urine concentrations of nitrate and nitrite normalized by creatinine excretion were significantly lower in women with preeclampsia compared with normal pregnant women (0.37 +/- 0.06 vs 0.69 +/- 0.11 micromol of nitrite per milligram creatinine, p. <0.05). CONCLUSIONS: Our study using concomitant measurement of plasma and urine nitrate and nitrite suggests a reduced production of nitric oxide in women with preeclampsia compared with normal pregnant women.     

Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide

OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis. DESIGN: Prospective, randomized, controlled study on anesthetized animals. SETTING: A cardiopulmonary research laboratory. SUBJECTS: Forty-seven male adult Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment. MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%). CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.     

Characterization of nitric oxide dependent changes in carbohydrate hepatic metabolism during septic shock

The role of nitric oxide in the alterations of liver carbohydrate metabolism during septic shock has been studied in fed and starved animals injected with bacterial lipopolysaccharide (LPS). One h after LPS injection an hyperglycemic peak was observed followed by hypoglycemia when the plasma nitric oxide concentration increased. However, in animals pharmacologically treated with nitric oxide donors only hypoglycemia was observed. In isolated hepatocytes from LPS treated rats an impairment of the gluconeogenic flux was observed accompanied by a decrease in the mRNA levels of the glucose transporter GLUT-2 and phosphoenolpyruvate carboxykinase, at the time that increased the mRNA levels of the inducible form of nitric oxide synthase. These results suggest that part of the effects observed in response to LPS challenge are due to early signaling molecules (cytokines and other factors molecules) whereas other effects can be attributed to nitric oxide synthesis which in turn has specific effects on hepatic metabolism.     

Involvement and dual effects of nitric oxide in septic shock

Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.     

Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels

Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (chi 2(1) = 16.04, P = .000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NOx levels, and measured genotype analysis showed that plasma NOx levels in those homozygous for the rare allele (64.9 +/- 7.8 mumol/L) were significantly higher (P = .000242) than those homozygous for the common allele (30.2 +/- 3.1 mumol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NOx levels (P = .0066). While many environmental factors have been shown to alter transiently plasma NOx levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NOx, i.e. basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.     

Hypotension does not alter the antinociceptive effect of nifedipine

We explored the relationship between antinociceptive and hypotensive effects of nifedipine (NIF) injected intraperitoneally ( ip, 15 mg/kg) and epidurally (epi, 20 microM), as compared to verapamil (VER, 10 mg/kg ip) and nitroglycerin (NTG, 0.1 and 0.15 mg/kg ip). The systolic blood pressure (BP) and tail-flick (TF) latencies were measured simultaneously every 10 min for 2 hours and individual values of both measurements were correlated. The highest antinociceptive as well as hypotensive effects were both measured in the group receiving NIF epi., with the correlation coefficient r2=0.2878. Injected ip., NIF revealed similar antinociceptive effect, whereas the other studied drugs were not effective. As to the degree of hypotensive activity, NIF epi was followed by VER, NTG 0.1, NIF ip. and NTG 0.15. No significant correlation was found between BP and TF latencies in any group receiving the drugs. We concluded that the antinociceptive response, measured by the tail-flick technique, is independent of the hypotensive activity of the studied drugs, including NIF.     

Establishment of patterned thalamocortical connections does not require nitric oxide synthase

Subplate neurons are early-generated neurons that project into the overlying neocortex and are required for the formation of ocular dominance columns. A subset of subplate neurons express nitric oxide synthase (NOS) and produce nitric oxide (NO), a neuronal messenger thought to be involved in adult hippocampal synaptic plasticity and also in the establishment of certain specific connections during visual system development. Here, we examine whether the NOS-containing subplate neurons are involved in ocular dominance column formation in the ferret visual system. Ocular dominance columns form in ferrets between postnatal day 35 (P35) and P60. NOS expression in the visual subplate is low at birth, increases to a maximum at the onset of ocular dominance column formation, and falls thereafter. Nevertheless, blockade of NOS with daily injections of nitroarginine from P14 to P56 fails to prevent the formation of ocular dominance columns, although NOS activity is reduced by >98%. To test further a requirement for NOS in the patterning of connections during CNS development, we examined the cortical barrels in the somatosensory system of mice carrying targeted disruptions of NOS that also received injections of nitroarginine; cortical barrels formed normally in these animals. In addition, barrel field plasticity induced by whisker ablation at birth was normal in nitroarginine-injected NOS knock-out mice. Thus, despite the dynamic regulation of NOS in subplate neurons, NO is unlikely to be essential for the patterning of thalamocortical connections either in visual or somatosensory systems.     

Arterial ketone body ratio does not correlate with ischemic changes during major hepatectomy

BACKGROUND/AIMS: The arterial ketone body ratio (AKBR) has been proposed as an accurate indicator of hepatic mitochondrial redox potential. However, recent studies of the utility of the AKBR as a biochemical marker have been called into question. It is not clear whether the AKBR is closely related to ischemic changes during major hepatectomy. METHODOLOGY: Arterial acetoacetate and beta-hydroxybutyrate concentrations were measured in eleven patients who underwent major hepatectomy. The ratio between them (AKBR) was calculated before and after vascular occlusion during the hepatectomy procedure. RESULTS: The AKBR increased following normothermic arterial or portal venous ischemia as compared to the levels prior to vascular occlusion in 36.4% of the patients who underwent major hepatectomy. An AKBR of less than 0.5 prior to vascular occlusion did not correlate with preoperative hepatocellular function. An AKBR of less than 0.7 throughout surgery was not a consistent risk factor for postoperative complications or liver dysfunction. CONCLUSIONS: The AKBR does not correlate with ischemic changes or postoperative complications after major hepatectomy.     

Effectiveness of nitric oxide inhalation in septic ARDS

STUDY OBJECTIVE: To evaluate the percentage of nitric oxide (NO) responders in septic shock patients with ARDS. Additionally, to investigate long-term NO effects on cardiac performance and oxygen kinetic patterns in NO responders vs nonresponders. DESIGN: Prospective cohort study. SETTING: ICU of a university hospital. PATIENTS: Twenty-five consecutive patients with a diagnosis of septic shock and established ARDS requiring inotropic and vasopressor support. INTERVENTIONS: After diagnosis of ARDS, NO was administered at 18 or 36 ppm. Patients demonstrating a NO-induced rise of arterial oxygen tension of 20% or more and/or a fall in mean pulmonary artery pressure of 15% or more were grouped as NO responders; others were grouped as nonresponders. MEASUREMENTS AND RESULTS: Ten patients (40%) were NO responders, while 15 patients (60%) were nonresponders. Mortality was 40% in NO responders and 67% in nonresponders (NS). NO responders developed a significantly lower mean pulmonary artery pressure (28 +/- 6 vs 33 +/- 6 mm Hg; p < 0.05), lower pulmonary vascular resistance (PVR: 258 +/- 73 vs 377 +/- 163 dyne.s.cm-5.m-2; p < 0.05), and higher PaO2/FIO2 ratio (192 +/- 85 vs 144 +/- 74 mm Hg; p < 0.05) within the study period. In responders, NO-induced afterload reduction resulted in increased right ventricular ejection fraction (RVEF: 40 +/- 7 vs 35 +/- 9%; p < 0.05), significantly higher cardiac index (CI: 4.5 +/- 1.1 vs 4.0 +/- 1.2 L.min-1.m-2; p < 0.05) and oxygen delivery (DO2: 681 +/- 141 vs 599 +/- 160 mL.min-1.m-2; p < 0.05) compared with nonresponders. In NO nonresponders, RVEF was correlated with PVR, CI, DO2, mixed venous oxygen saturation (SvO2), and oxygen extraction ratio (O2ER) (r = +/- 0.60 to +/- 0.69; p < 0.05). No significant correlation between RVEF and any of these parameters was observed in responders. SvO2 (75 +/- 7 vs 69 +/- 8%; p < 0.05) and O2ER (0.24 +/- 0.06 vs 0.27 +/- 0.06; p < 0.05) were significantly different between responders and nonresponders, while no difference in oxygen consumption was observed (161 +/- 41 vs 153 +/- 43 mL.min.m-2). CONCLUSIONS: Inhaled NO is effective in only a subgroup of septic ARDS patients, with a higher, but insignificantly different percentage of survivors in the responder group. NO responders were characterized by increased RVEF accompanied by higher CI, DO2, and lower O2ER. In nonresponders, RVEF remained depressed, with a close correlation between RVEF and CO as well as DO2 and O2ER. Thus, nonresponders seem to suffer from impaired cardiac reserves and correspondingly lower oxygen transport variables.     

Sertraline treatment does not increase plasma prolactin levels in healthy subjects

Peliosis is a rare disease that is characterized by multiple blood-filled cystic spaces. We report the computed tomographic findings of splenic rupture secondary to splenic peliosis in a patient receiving anabolic steroids for aplastic anemia.     

Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise

This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressure, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-L-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46 +/- 23, 129 +/- 44, and 63 +/- 32 nmol/min at each speed respectively, P < .05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.     

Measurement of nitric oxide in the rat cerebral cortex during hypercapnoea

Changes in nitric oxide (NO) concentration and cerebral blood flow (CBF) in the parietal cortex during hypercapnoea were investigated in anaesthetized rats, using a NO-selective electrode and laser Doppler flowmetry. When hypercapnoea was induced by inhalation of 5% CO2 for 10 min, both the NO concentration and CBF increased. After administration of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, both the basal NO and CBF decreased, and responses to hypercapnoea were also significantly suppressed by 70.1% and 73.2%, respectively, compared with the control state. These results suggest that NO derived from nNOS is involved not only in maintaining resting cerebral circulation but also in regulating CBF response during hypercapnoea.     

Long-term cardiovascular role of nitric oxide in conscious rats

Twenty-eight non-allergic subjects suffering from mono- and bilateral chronic nasal respiratory obstruction underwent investigation employing routine semeiological examination. The real cause of the obstruction, however, was not ascertained. The subjects underwent nasal endoscopy employing rigid fiberoptics and, on the grounds of the findings, a cat scan of the nasal fossae in coronal projection, was made. The results of the study revealed involvement of the middle meatal area in 63% of bilateral nasal obstructions (B.N.O.) and in 77.7% of the monolateral obstructions (M.N.O.). Choanal pathology was evident in 21% of the B.N.O., while isolated upper and posterior deviations of the septum were present in 23.6% of the B.N.O. and in 22.5% of the M.N.O. Meatal involvement was due to the presence of anatomic changes of the middle turbinate (m.t.) such as concha bullosa, paradoxical deviation of the m.t. and of the uncinate process. Moreover, small "mucosal polyps" were also noticed in this meatal area. Cat scans, performed on 18 of these 28 patients, confirmed and defined meatal alterations found with endoscopy in 16 cases. In the remaining 2 cases, cat scans showed a thickening of the mucosal+bony tissues of the m.t. The Authors did not consider CT necessary in 10 subjects in that endoscopy sufficed in explaining obstruction symptoms.     

Circulating methemoglobin and nitrite/nitrate concentrations as indicators of nitric oxide overproduction in critically ill children with septic shock

OBJECTIVES: To examine the relationship between circulating methemoglobin and nitrite/nitrate concentrations and to compare these markers of nitric oxide overproduction with clinical variables in children diagnosed with septic shock. DESIGN: Prospective, controlled, clinical study. SETTING: Pediatric intensive care unit and outpatient clinic in a children's hospital. PATIENTS: Twenty-two children diagnosed with septic shock and ten age-matched healthy control patients. INTERVENTIONS: Patients diagnosed with septic shock had blood specimens taken on study entry and every 6 hrs for 72 hrs for methemoglobin and nitrite/nitrate determinations. Single blood specimens were obtained from controls. MEASUREMENTS AND MAIN RESULTS: Circulating methemoglobin and nitrite/nitrate concentrations were significantly higher in children diagnosed with septic shock in comparison with healthy control children (p = .01 and .05, respectively). Peak nitrite/nitrate concentrations correlated with serum creatinine (r2 = .19; p = .04) and were inversely correlated with arterial pH (r2 = .28; p = .01) and urine output (r2 = .21; p = .03) when analyzed by log-linear regression. There were no significant relationships between methemoglobin and nitrite/nitrate or between methemoglobin and any other clinical variable. CONCLUSIONS: Circulating methemoglobin and nitrite/nitrate concentrations are increased in children diagnosed with septic shock. Plasma nitrite/nitrate values correlate with selected clinical variables in these children. Circulating methemoglobin measurements are not superior to plasma nitrite/nitrate concentrations as an indicator of endogenous overproduction of nitric oxide in children diagnosed with septic shock. A need remains to develop markers of endogenous nitric oxide activity that have greater accuracy and reliability.     

Multiple nitric oxide sources in neurogenic plasma extravasation in rat hindpaw skin

The contribution of nitric oxide (NO) to capsaicin-evoked plasma extravasation was studied in rat hindpaw skin. Two inhibitors of NO synthase were used: 7-nitroindazole, with a selectivity for nerve-derived NO, and the L-arginine derivative, N(omega)-nitro-L-arginine (L-NOARG), which is a non-selective inhibitor. Plasma extravasation was induced by intraplantar injection of 5 microg/50 microl capsaicin and measured by the Evans blue leakage technique. Both acute and chronic administration of 7-nitroindazole significantly reduced capsaicin-evoked plasma extravasation in rat hind-paw skin, whereas L-NOARG enhanced it. This enhancement was abolished non-stereospecifically by either L- or D-arginine. Our results suggest that NO production from different sources yields a complex action in maintaining the endothelial integrity in neurogenic plasma extravasation.     

Lymph node size does not correlate with the presence of prostate cancer metastasis

Prostate carcinoma (PC) is the second leading cause of cancer death in men in the western world. Although the role of oncogenes and growth factors in prostate carcinoma is still unclear, overexpression of the epidermal growth factor receptor (erbB-1) and the proto-oncogene erbB-2 have been reported in prostate tumors, and erbB-2 related to poor prognosis and distant metastasis. Recent allelotyping studies in prostate cancer have shown chromosomal gains in 7p and 17q, regions where erbB-1 and erbB-2 are localized respectively, although no direct evidence of an increased gene copy number of either erbB-1 or erbB-2 has been reported. To address this question, we analyzed 20 benign prostatic hyperplasia (BPH) samples and 36 samples of metastatic and non-metastatic PC by means of semiquantitative PCR. Thus, 64% (11/17) and 52% (10/19) of metastatic and non-metastatic tumors respectively showed gains of the relative genomic content of erbB-1 and an association of erbB-1 with prostate cancer but not with metastasis. Additionally, 41% (7/17) of metastatic samples showed gains of erbB-2 genomic content, suggesting an association of erbB-2 with metastasis and poor prognosis (p<0.005). No gains of erbB-1 or erbB-2 genomic content were detected in the BPH samples.     

Inhibition of nitric oxide synthesis does not improve interleukin-2-mediated antitumor effects in vivo

BACKGROUND: Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.     

Metabolism of agmatine into urea but not into nitric oxide in rat brain

Agmatine is a guanidino compound abundant in bacteria and plants where it serves as a precursor for polyamine synthesis. It can interfere with several neurotransmission-related functions and can exert neuroprotective effects after brain injury. Agmatine was recently identified in mammalian brain and its synthesis by arginine decarboxylation was characterized. Its metabolism by the brain is, however, unknown. Here we report evidence indicating that agmatine can be selectively metabolized in the rat brain (cerebellum) into urea and thus, may lead to formation of putrescine, the precursor of polyamine synthesis. In addition, while agmatine can inhibit brain nitric oxide synthase, it did not serve as a substrate for nitric oxide formation.