NO Metabolite Levels Research Articles

Expression of inducible nitric oxide synthase in primary culture of rat bladder smooth muscle cells by plasma from cyclophosphamide-treated rats

Intraperitoneal administration of cyclophosphamide (50–150 mg/kg) for 6 or 12 h induced edema and hemorrhagic changes in rat bladder, which were both dose and time-dependent. Pretreatment with nitric oxide synthase (NOS) inhibitors N G-nitro- l-arginine methyl ester ( l-NAME, 40 mg/kg) or with s-methylisothiourea (40 mg/kg) ameliorated the cyclophosphamide-induced cystitis. Cyclophosphamide administration also produced increases in NO-metabolite levels (nitrate+nitrite) in the urine and plasma of rats. Greater increases in NO metabolites were observed with 150 than with 50 mg/kg of cyclophosphamide, and at 12 than at 6 h after cyclophosphamide injection. Pretreatment with l-NAME and s-methylisothiourea significantly reduced cyclophosphamide-induced increases in urine and plasma NO-metabolite levels. To explore the mechanism by which cyclophosphamide increases the expression of inducible NOS (iNOS), primary cultures of rat bladder smooth muscle were developed. Exposure to tumor necrosis factor α (TNF-α) plus interferon γ, produced a marked increase in the expression of iNOS and in NO production in the culture medium. However, exposure to cyclophosphamide or to its metabolite acrolein (10 −6–10 −4 M for 24 h) did not increase iNOS or NO-metabolite levels. On the other hand, incubation of primary cell cultures with plasma from rats treated with cyclophosphamide (150 mg/kg, 12 h) produced a marked increase in iNOS expression and NO production. Taken together, our results indicate that NO plays an important role in the pathogenesis of cyclophosphamide-induced cystitis in rats, and some factors may be released in cyclophosphamide-treated rat plasma which stimulate iNOS expression in primary culture of rat bladder smooth muscle cells.

Increased plasma tetrahydrobiopterin in septic shock is a possible therapeutic target

Hemoperfusion with a column of polymyxin B immobilized on fibers (PMX) has been used to adsorb endotoxin in-patients with septic shock. PMX hemoperfusion (PMX-H) increases blood pressure (BP) too rapidly for the effect to be attributable to endotoxin removal. Since inducible NO synthase (iNOS) is known to be involved in the profound hypotension, we hypothesized that a decrease of tetrahydrobiopterin (BH 4), an essential cofactor of iNOS, might account for the rapid effect of PMX-H on BP, if plasma BH 4 is increased concomitantly with NO in septic shock patients and if PMX can decrease BH 4. BH 4 was evaluated by measuring total biopterin, which can include derivatives of BH 4 by using high-performance liquid chromatography (HPLC). We confirmed that PMX fabric time dependently decreased total biopterin concentration in vitro. The plasma level of total biopterin in shock patients was indeed markedly elevated compared with that in volunteers (131.1±33.4 vs. 10.4±1.1 nM, n=5, P<0.01). Level of NO metabolites (NOx) were 173.9±104.7 versus 28.7±11.6 μM ( P<0.01). In beagles, plasma total biopterin was 44.7±6.9 nM at baseline, reached 118±28.6 nM after lipopolysaccharide (LPS) injection, and fell to 70.2±15.8 nM after PMX-H. Plasma NOx concentration was increased from 15.2±4.2 to 41.0±7.5 μM by LPS treatment. BP was 130±11.3 mmHg at baseline, 82.2±8.3 mmHg at 14 h after LPS, and 115.2±16.0 mmHg after PMX-H. In rats, plasma total biopterin was 88.8±1.5 nM at baseline, 383.7±144.2 nM after LPS and 177.0±14.2 nM after PMX-H. Plasma NOx was also increased after LPS (from 34.6±4.4 to 1445.6±376.0 nM). The marked increase in total biopterin concomitantly with NOx in septic shock patients and its reduction by PMX-H in animal models of septic shock are consistent with our hypothesis, and appear to justify further research on BH 4 removal as a potential therapeutic target.

Effect of sepsis and cardiac surgery with cardiopulmonary bypass on plasma level of nitric oxide metabolites, neopterin, and procalcitonin: correlation with mortality and postoperative complications

To examine the hypothesis that nitrite/nitrate, neopterin, and procalcitonin (PCT) levels can be useful predictors of sepsis-associated mortality and predictors of the postoperative complications after cardiopulmonary bypass (CPB). Prospective clinical study. Intensive care unit of the Medical University Hospital. 41 patients with sepsis, 42 patients subjected to open heart surgery with CPB, and 30 healthy volunteers. Nitrite/nitrate, neopterin, and PCT levels were measured in septic patients as soon as sepsis was recognized and then on the 2nd, 3rd, and 5th days of treatment. Statistically significant differences between survivors and nonsurvivors were found for neopterin and PCT. The area under receiver operating characteristic curve (AUC) for both parameters as predictors of mortality was above 0.8. The nitrite/nitrate level was also higher in nonsurvivors, but the AUC remained below 0.8, which indicates poor predictive power. The same parameters were measured in patients undergoing cardiac surgery before, during and after CPB establishment. The development of post-operative complications was correlated with increased postoperative neopterin and PCT levels. Additionally, neopterin was found as an early marker for the prognosis of postoperative complications, since patients who developed organ dysfunction had had elevated concentration of this parameter even before surgery (AUC 0.83). Measurement of NO metabolite levels was less specific and less sensitive. Our results confirm the value of PCT and neopterin measurement as diagnostic tools in monitoring the clinical course of patients in intensive care units.

Effect of Strenuous Arm Exercise on Oxidized-LDL-Potentiated Platelet Activation in Individuals with Spinal Cord Injury

This study investigates how strenuous arm exercise affects oxidized-low density lipoprotein (O(X)-LDL) mediated-platelet activation in patients with SCI. Ten patients with SCI and ten age- and sex-matched healthy subjects exercised strenuously using an arm crank ergometer. The following measurements were taken both when the subjects were at rest, and immediately after exercise: plasma lipid profile, O(X)-LDL mediated platelet aggregability and [Ca(2+)]i, urinary 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 8-iso-prostaglandin F(2alpha), (8-iso-PG F(2alpha)) contents, and plasma NO metabolite (nitrite plus nitrate) level. Based on these measurements, the major findings of this study can be summarized as follows: 1) the SCI group had higher urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB2 contents, but a lower plasma nitrite plus nitrate level than the control group; 2) at rest, the SCI group had a higher platelet aggregability and [Ca(2+)]i, and O(X)-LDL-potentiated platelet activation than the control group; 3) O(X)-LDL-potentiated platelet aggregation was enhanced by strenuous arm exercise in both groups, but the effect of exercise was more pronounced in the SCI group than in the control group; 4) treating the platelet with L-arginine inhibited O(X)-LDL-potentiated platelet activation in both groups. The study concludes that individuals with SCI had more extensive resting and exercise-enhanced O(X)-LDL-potentiated platelet activation and greater amounts of preformed lipid peroxides than those without SCI. Therefore, supplementation therapy with antioxidants may be needed for patients with SCI, especially in a strenuous arm exercise period.

Role of Nitric Oxide and Reactive Oxygen Species in Cyclosporin-Induced Hypertension in Transplanted Patients.

637 Hypertension is a major side effect of Cyclosporin (CsA). While mechanism/s responsible are unclear, CsA-induced endothelial dysfunction and CsA-induced hypertension has been attributed to CsA effect on the endothelial-derived factors controlling vasomotor tone. Endothelial NO is crucial in the manteinance of a state of basal vasodilation and recently an NO mediated counterregulatory mechanism protective from CsA-induced vasoconstriction has been suggested. Our study evaluates ecNOS gene status (Polymerase Chain Reaction analysis) and plasma level of NO metabolites (NO2−/NO3−) (ELISA) in 10 heart (HT) and 10 kidney (KT) transplanted patients under chronic CsA treatment with CsA-induced hypertension. Diagnosis of CsA-induced hypertension in HT was based on the development of hypertension only after CsA administration and without improvement after the tapering of steroids associated to CsA. In KT, by the development of hypertension only after transplant, for those normotensive before the transplant, and by the worsening of a preexisting hypertension notwithstanding the tapering of steroids associated to CsA treatment. Since CsA increases superoxide anions, which combine with NO to yield peroxynitrite, a powerful oxidant, plasma peroxynitrites and hydroperoxides were evaluated (HPLC) as index of the presence of superoxides and free radicals and therefore of oxidative stress. 10 healthy subjects were used as controls (C). Quantification of monocyte ecNOS mRNA from patients and controls demonstrated NO system up regulation in patients notwithstanding the hypertension. The mean ecNOS to β-actin ratio was 1.80±0.85 in patients vs 0.40±0.09 in C (p<0.04). (NO2−/NO3−) plasma level was 34.03±14.32 mM in patients vs 11.53±5.64, p<0.001. Hydroperoxides were also increased in patients vs C: 3.4±1.4 i.a.u. vs 1.3±0.6, p<0.007 (from colesterol esters) and 10.6±6.4 vs 1.3±0.8, p<0.008 (from triglycerides) as well as peroxynitrite plasma level (0.32±0.11 mM/L vs undetectable in C. This study confirms a CsA induced NO system up regulation in transplanted patients. However, the counterregolatory system to CsA-induced vasoconstriction could be canceled in patients by CsA induced superoxides and free radicals production which, increasing NO metabolism and therefore reducing its efficacy as vasorelaxing factor could contribute to CsA induced vasoconstriction and hypertension and promoting remodelling processes, predispose to long term hypertensive complications such as cardiac hypertrophy and atherosclerosis.

P–31 - Plasma levels of NO metabolites in spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and malignant and precocious SHRSP (M-SHRSP)

P–31 - Plasma levels of NO metabolites in spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and malignant and precocious SHRSP (M-SHRSP)

Interleukin 1beta-stimulated production of nitric oxide in rat hepatocytes is mediated through endogenous synthesis of interferon gamma.

The multiple interlocking regulatory mechanisms that underlie induction of hepatocyte inducible nitric oxide synthase (iNOS) expression are largely unknown. Although previous work has indicated the requirement for multiple proinflammatory cytokines to induce hepatocyte NO production, investigators have recently shown that interleukin-1beta (IL-1beta) alone can initiate iNOS expression. In contrast, interferon gamma (IFN-gamma) serves as the sole initiating factor in other cell systems. On the basis of the known ability of IL-1beta to induce transcription and translation of the IFN family of genes, we hypothesized that IL-1beta-mediated hepatocyte expression of iNOS is dependent on endogenous IFN-gamma synthesis. In a system of rat hepatocytes in primary culture, IL-1beta induced production of both NO and IFN-gamma. Using in situ hybridization and immunoblot analysis, IFN-gamma messenger RNA (mRNA) and protein were detected in hepatocytes exposed to IL-1beta. Inhibition of NO synthesis using the competitive substrate inhibitor N-monomethyl-L-arginine (100 micromol/L) did not alter the extent of IL-1beta-mediated IFN-gamma synthesis. In contrast, anti-IFN-gamma antibody or inhibition of IFN-gamma mRNA translation by addition of antisense IFN-gamma oligodeoxynucleotide probes resulted in undetectable levels of NO metabolites and iNOS protein. Repletion of IFN-gamma to the system restored NO production to levels noted in the presence of IL-1beta alone. Transient transfection analysis using a rat hepatocyte iNOS promoter-reporter gene plasmid construct showed that IL-1beta-induced promoter activation was abolished in the presence of anti-IFN-gamma or antisense IFN-gamma. Again, addition of IFN-gamma to the system restored activity of the iNOS promoter. Parallel experiments examining IL-1beta-mediated endogenous hepatocyte IL-1beta and TNF-alpha synthesis indicated no role for these cytokines in the induction of iNOS expression by IL-1beta. It is concluded that IL-1beta-mediated hepatocyte synthesis of NO is dependent on the simultaneous endogenous synthesis of IFN-gamma.