Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse

1. The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse. 2. Administration of endotoxin (E. Coli, 026:B6, 12.5 mg kg(-1), i.v.) elevated the concentration of tumour necrosis factor-alpha (TNF-alpha) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin-6 (IL-6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study. 3. Endotoxin (12.5 mg kg(-1), i.v.) induced the expression of a Ca2+-independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period. 4. The vasopressor effect of noradrenaline (0.5-4 microg kg(-1) min(-1), i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg(-1), i.v). 5. The NO synthase inhibitor NG-monomethyl-L-arginine HCl (L-NMMA; 1-10 mg kg(-1), i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg(-1), i.v.). 6. In an attempt to maintain a constant blood concentration, L-NMMA was administered as a continuous infusion (10 mg kg(-1) h(-1), i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg(-1), i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation. 7. The fall in blood pressure following endotoxin (3 mg kg(-1), i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase. 8. In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.     

In vivo quantification of endotoxin-induced nitric oxide production in pigs from Na15NO3-infusion

1. In this investigation the NO production rate is quantified in the pig during normotensive endotoxin-induced shock with increased cardiac output and during subsequent treatment with the NO synthase inhibitor N omega-monomethy-L-arginine (L-NMMA). NO production rate was derived from the plasma isotope-enrichment of 15N-labelled nitrate (15NO3-). 2. Three groups of animals (control, n = 5; endotoxin, n = 6; endotoxin + L-NMMA, n = 6) were anaesthetized and instrumented for the measurement of systemic and pulmonary haemodynamics. Each animal received a primed-continuous infusion of stable, non-radioactively labelled Na15 NO3 (bolus 30 mg, infusion rate 2.1 mg h-1). Arterial blood samples were taken 5, 10, 15, 30, 60 and 90 min later and every 90 minutes until the end of the experiment. 3. Continuous i.v. infusion of endotoxin was incrementally adjusted until mean pulmonary artery pressure (PAP) reached 50 mmHg and subsequently titrated to keep mean PAP approximately 35 mmHg. Hydroxyethylstarch was administered as required to maintain mean arterial pressure (MAP) > 60 mmHg. Six hours after the start of the endotoxin continuous i.v. L-NMMA (1 mg kg-1 h-1) was administered to the endotoxin + L-NMMA group. Haemodynamic data were measured before as well as 9 h after the start of the endotoxin. 4. After conversion of NO3- to nitro-trimethoxybenzene and gas chromatography-mass spectrometry analysis the total NO3- pool, basal NO3- production rate and the increase per unit time in NO3- production rate were calculated from the time-course of the 15NO3- plasma isotope-enrichment. A two compartment model was assumed for the NO3- kinetics, one being an active pool in which newly generated NO3- appears and from which it is eliminated, the other being an inactive volume of distribution in which only passive exchange takes place with the active compartment. 5. Although MAP did not change during endotoxin infusion alone, cardiac output (CO) increased by 42 +/- 40% (P < 0.05 versus baseline) by the end of the experiment due to a significant (P < 0.05 versus baseline) fall in systemic vascular resistance (SVR) to 65 +/- 25% of the baseline value. L-NMMA given with endotoxin did not change MAP, and both CO and SVR were maintained close to the pre-shock levels. 6. Baseline plasma NO3- concentrations were 43 +/- 13 and 40 +/- 10 mumol l-1 in the control and endotoxin animals, respectively, and did not differ at the end of the experiment (39 +/- 8 and 44 +/- 15 mumol l-1, respectively). The mean NO3- pool and basal NO3- production rate were 1155 +/- 294 mumol and 140 +/- 32 mumol h-1, respectively, without any intergroup difference. Endotoxin significantly increased NO3- production rate (23 +/- 10 mumol h-2, P < 0.05 versus control (6 +/- 7 mumol h-2) and endotoxin + L-NMMA groups). L-NMMA given with endotoxin (-1 +/- 2 mumol h-2, P < 0.05 versus control and endotoxin groups) had no effect. 7. Analysis of the time course of the 15NO3- plasma isotope enrichment during primed-continuous infusion of Na15NO3 allowed us to quantify the endotoxin-induced increase in NO3- production rate independently of total NO3- plasma concentrations. Low-dose L-NMMA blunted the increase in NO3- production rate while maintaining basal NO3- formation.     

Induction of nitric oxide production by polyosides from the cell walls of Streptococcus mutans OMZ 175, a gram-positive bacterium, in the rat aorta

The cardiovascular dysfunctions associated with septic shock induced by gram-negative or gram-positive bacteria (gram-positive or gram-negative septic shock) are comparable. In gram-negative septic shock, lipopolysaccharide (LPS) induces nitric oxide (NO) synthase, which contributes to the vascular hypotension and hyporeactivity to vasoconstrictors. The role of NO in gram-positive septic shock and the nature of the bacterial wall components responsible for the vascular effects of gram-positive bacteria are not well known. This study investigated the vascular effects of cell wall serotype polyosides, rhamnose glucose polymers (RGPs), from Streptococcus mutans, in comparison with lipoteichoic acid (LTA) from Staphylococcus aureus, on the induction of NO synthase activity in the rat aorta. We show that 10 microg of both RGPs and LTA per ml induced hyporeactivity to noradrenaline, L-arginine-induced relaxation, increases of 2.2- and 7.8-fold, respectively, of cyclic GMP production, and increases of 7- and 12-fold in nitrite release. All of these effects appeared after several hours of incubation and were inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Electron paramagnetic resonance spin trapping experiments demonstrated directly that RGPs and LTA induced NO overproduction (four- to eightfold, respectively) in rat aortic rings; this production was inhibited by L-NAME and prevented by dexamethasone. These results demonstrate directly the induction of NO production in vascular tissue by LTA and show that another, chemically different component of gram-positive bacteria can also have these properties. This result suggests that different components of the gram-positive bacterial wall could be implicated in the genesis of cardiovascular dysfunctions observed in gram-positive septic shock.     

Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition

1. This study investigated the effects of low dose endotoxin (lipopolysaccharide, LPS) on (i) systemic haemodynamics, (ii) renal blood flow (RBF), (iii) renal cortical and medullary perfusion and (iv) renal function in the anaesthetized rat. We have also investigated the effects of nitric oxide (NO) synthase (NOS) inhibition with NG-methyl-L-arginine (L-NMMA) on the alterations in systemic and renal haemodynamics and renal function caused by endotoxin. 2. Infusion of low dose LPS (1 mg kg-1 over 30 min, n = 6) caused a late fall in mean arterial blood pressure (MAP, at 5 and 6 h after LPS), but did not cause an early (at 1-4 h after LPS) hypotension. The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Infusion of L-NMMA (50 micrograms kg-1 min-1 commencing 60 min before LPS and continued throughout the experiment, n = 7) abolished the delayed hypotension and significantly attenuated the vascular hyporeactivity to NA (at 2-6 h). 3. Infusion of LPS (1 mg kg-1 over 30 min, n = 6) caused a rapid (within 2 h) decline in renal function (measured by inulin clearance) in the absence of a significant fall in MAP or renal blood flow (RBF). L-NMMA (n = 7) attenuated the impairment in renal function caused by LPS so that the inulin clearance in LPS-rats treated with L-NMMA was significantly greater than in LPS-rats treated with vehicle (control) at 3-6 h after infusion of LPS. 4. Endotoxaemia also caused a significant reduction in renal cortical, but not medullary perfusion (measured as Laser Doppler flux). Infusion of L-NMMA caused a significant further fall in cortical perfusion and a significant fall in medullary perfusion in the absence of changes in RBF. 5. Infusion of LPS resulted in a progressive increase in the plasma levels of nitrite/nitrate (an indicator of the formation of NO), so that the plasma concentration of nitrite/nitrate was significantly higher than baseline at 150 to 330 min after LPS. Infusion of L-NMMA attenuated the rise in the plasma concentration of nitrite/nitrate (at 270 and 330 min, P < 0.05) caused by LPS. 6. Thus, the renal dysfunction caused by injection of low dose of endotoxin in the rat occurs in the absence of significant falls in blood pressure or total renal blood flow. Inhibition of NOS activity with L-NMMA attenuates the renal dysfunction caused by endotoxin (without improving intrarenal haemodynamics), suggesting that an overproduction of NO may contribute to the development of renal injury and dysfunction by causing direct cytotoxic effects.     

N(G)-monomethyl-L-arginine improves survival in a pig model of abdominal sepsis

OBJECTIVE: To test the effect of a continuous infusion of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on survival rate and hemodynamics in a pig model of endogenous peritoneal live bacterial sepsis. DESIGN: Prospective, randomized trial. SETTING: Laboratory at a university medical center. SUBJECTS: Thirty-five pigs with an average weight of 26 kg (range 21 to 33). INTERVENTIONS: After surgical preparation, animals (control, n=6) given anesthesia and fluids were observed for 9 hrs. Fifteen experimental animals received 0.5 g of cecal content/kg of body weight intraperitoneally after surgery. Nine of these animals received standard anesthesia and fluids and were observed for 9 hrs or until death. Six animals received a continuous infusion of L-NMMA (10 mg/kg/hr) 3 hrs after sepsis induction. Starting 3 hrs after surgery, five nonrandomized animals were given anesthesia and fluids and received a 6-hr continuous infusion of L-NMMA (10 mg/kg/hr). An additional nine animals were anesthetized and blood samples were taken to determine plasma nitrate concentrations in nonoperated pigs. MEASUREMENTS AND MAIN RESULTS: L-NMMA treatment increased 9-hr survival in septic animals from 11% to 83% (p < .001), prevented a further decrease in mean arterial pressure and restored mean arterial pressure to control levels (p < .00002 vs. nontreated septic animals). Mean pulmonary arterial pressure increased slightly during L-NMMA infusion (p < .0003). Coronary blood flow was preserved during L-NMMA treatment. Cardiac index and urine production reached and maintained control levels during L-NMMA treatment of septic animals. Mean central venous pH did not deteriorate during L-NMMA treatment. Animals treated with L-NMMA had plasma nitrate concentrations similar to nonseptic control animals. The results from the nonseptic control group receiving L-NMMA suggest that a substantial part of the effect of L-NMMA in this model of septic shock may be due to inhibition of the constitutive nitric oxide production. CONCLUSIONS: In this porcine model of peritoneal sepsis, infusion of L-NMMA increased survival rate and maintained mean arterial pressure without worsening tissue oxygenation. Coronary blood flow, cardiac index, systemic vascular resistance, and urine production were well maintained during L-NMMA treatment.     

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.     

MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies

Septic shock is a major cause of death among patients in intensive care units. It has a mortality rate of 20% to 80%. The clinical syndrome of septic shock is characterised by hypotension, hyporesponsiveness to vasoconstrictors and volume depletion which will then lead to multiorgan dysfunction and death. Except for surgical and supportive care, no specific therapy is known. Recently interest has been focused on the role of nitric oxide (NO) in septic shock. Large amounts of NO released by the endothelium and vascular smooth muscle cells lead to profound vasodilation and hyporesponsiveness to vasoconstrictors. The cytotoxic effect of NO could also cause tissue injury and organ failure. Inhibition of NO synthase, the enzyme responsible for NO production, has been proposed as a new therapy for septic shock. However, experimental reports have provided conflicting results, demonstrating both beneficial and detrimental effects. A brief review of the role of NO in septic shock and the possible use of NO synthase inhibitors as potential therapeutic agents is presented here.     

Prevention of endotoxin shock by an antibody against leukocyte integrin beta 2 through inhibiting production and action of TNF

Septic shock remains a serious disorder associated with high mortality. Accumulating evidence indicates that TNF is a major and essential mediator of endotoxin shock. We report here that administration of an antibody against CD18 dramatically reduced endotoxin-induced shock in rabbits as revealed by prevention of severe hypotension, metabolic acidosis and a pathological change suggestive of disseminated intravascular coagulation with concomitant inhibition of elevation of plasma TNF activity. The anti-CD18 antibody also inhibited the hypotension induced by administering recombinant TNF. Furthermore, an antibody against a ligand for CD18 complexes, intercellular adhesion molecule-1, also prevented TNF-induced shock as well as endotoxin shock in rabbits. These observations suggest that adhesion of leukocytes to endothelium may be of primary importance in the action of TNF as well as in the production of TNF in vivo and that the antibody against adhesion molecules could be of therapeutic benefit in life-threatening septic shock in humans.     

Endogenous vasopressin increases acute endotoxin shock-provoked gastrointestinal mucosal injury in the rat

Administration of a low dose of endotoxin (from Escherichia coli, 3 mg kg(-1), i.v.), which does not affect vascular permeability or blood pressure over 1 h, leads to the release of endogenous vasopressin and damage to the mucosal microvasculature. Thus, endogenous vasopressin could be involved in septic shock. In the present study, we investigated the role of endogenous vasopressin in gastrointestinal mucosal injury induced by acute endotoxin shock, which was generated in rats by administering a high dose of E. coli endotoxin (50 mg kg(-1), i.v.). Tissues were removed 15 min after endotoxin. The vasopressin V1 receptor antagonist, d[CH2]5Tyr[Me]arginine-vasopressin (0.2-1 microg kg(-1), i.v.), was injected 10 min before endotoxin. Monastral blue (30 mg kg(-1), i.v.), which stains damaged vasculature, was injected 10 min before autopsy. Endotoxin reduced systemic arterial blood pressure (from 115+/-5 to 42+/-4 mmHg), generated macroscopic and microvascular injury, and elevated plasma vasopressin levels (from 3.4+/-0.2 to 178+/-16 pg ml(-1)). The vasopressin V1 receptor antagonist reduced this macroscopic injury, and in the vasopressin-deficient Brattleboro rat a similar reduction of gastrointestinal mucosal damage was found. Substantial decreases in endotoxin-induced microvascular damage were observed in each tissue, e.g., the gastric Monastral blue staining was reduced by 47+/-3% and 96+/-3% (P < 0.01) after vasopressin V1 receptor antagonist treatment and in Brattleboro rats, respectively. Vasopressin, acting through its V1 receptors, thus appears to be involved in acute endotoxin shock-provoked gastrointestinal injury.     

Central administration of saikosaponin-d increases corticotropin-releasing factor mRNA levels in the rat hypothalamus

The free radicals nitric oxide (.NO) and superoxide (O2-) are known to react to form peroxynitrite (ONOO-), a highly reactive species. Peroxynitrite has been suggested to play an important role in the cellular damage associated with the overproduction of .NO, but there are very limited data regarding its in vivo formation. Here we demonstrate that injection of endotoxin into rats leads to the expression of an inducible isoform of .NO synthase (iNOS) in the thoracic aorta at 6 h and an increase in the circulating levels of nitrite/nitrate. Moreover, at the same time point, there is a marked increase in the immunoreactivity of nitrotyrosine, a marker of peroxynitrite in the aorta. The formation of nitrotyrosine was prevented by inhibiting the activity of NOS by NG-methyl-L-arginine in vivo. Our data suggest that during endotoxin shock, part of .NO, produced following the induction of iNOS, is converted into peroxynitrite in the vicinity of large blood vessels. The demonstration of the in vivo formation of peroxynitrite at sites of .NO overproduction may necessitate the development of novel and additional approaches for limiting or preventing .NO-related cytotoxic or vasodilatory actions during circulatory shock.     

Platelet and brain alpha 2-adrenoceptors and cardiovascular sensitivity to agonists in dogs suffering from endotoxic shock

We examined the changes in alpha 2-adrenoceptor binding on platelet and brain membranes of dogs treated with a non-lethal dose of endotoxin (0.1 mg/kg intravenously), and the alpha 2-adrenoceptor mediated cardiovascular effects during endotoxin shock. At 2 h, 24 h, and 7 days after endotoxin administration, the number of binding sites (Bmax) of [3H]yohimbine binding decreased and equilibrium dissociation constants (Kd) increased in platelets, whereas both Bmax and Kd decreased in either cerebral cortex or medulla oblongata. After 30 days of endotoxin administration, there were no significant differences in Bmax or Kd between the treated and untreated animals in both platelets and brain tissues. Significant positive correlations were observed for Bmax values between platelets and brain tissues, although negative correlations for Kd values between platelets and brain were not significant. Significant negative correlations were also observed between plasma catecholamine concentrations and platelet alpha 2-adrenoceptor number, and between plasma noradrenaline and medulla alpha 2-adrenoceptor number. Pretreatment with E coli endotoxin diminished cardiovascular effects such as bradycardia, hypotension, and increase in systemic vascular resistance induced by either i.v. clonidine or xylazine. This suggests that alpha 2-adrenoceptor activity is impaired in the central nervous system as well as in the peripheral vascular system during endotoxin shock. Therefore, platelets may in part represent a good model which reflects the alpha 2-adrenoceptor changes in the central nervous system and peripheral vascular system during and after endotoxin shock.     

Overproduction of nitric oxide in pathophysiology of blood vessels

Sustained production of large amounts of nitric oxide (NO) is induced in blood vessels by inflammatory stimuli as a result of the expression of the inducible form of NO-synthase (NOS-2). This happens in systemic inflammatory reactions like septic shock and in local reactions produced by endothelium denudation and atherosclerosis. NOS-2 activity in blood vessels may protect tissues by virtue of the vasodilating, anti-thrombotic and leukocyte adhesion inhibitory effects of NO. It may also participate in vascular remodeling as a result of the antiproliferative and pro-apoptotic actions of NO. However excessive production of NO in blood vessels is involved in circulatory failure that takes place in systemic inflammatory reactions and it may be cytotoxic for surrounding tissues. For these reasons, inhibition of NO overproduction has been proposed in the treatment of septic shock. Selective inhibitors of NOS-2 activity or NO trapping agent, or both, might prove to be valuable drugs in the treatment of some inflammatory diseases. The conditions in which NO shifts from a tissue protective to a damaging role are not well elucidated. Recent findings suggest that the interactions with superoxide radicals, thiols, and metals (particularly with Fe2+) may be important not only in buffering excess NO produced by NOS-2, but also in channeling it from physiologically to pathophysiologically relevant targets. It has also been found recently that adventitial cells may play an important part in vascular NO production and generation of NO stores in the media layer. The ultimate effect of NO in blood vessels might depend on its site of production, local concentration, and interactions with other tissue components.     

Evaluation of the dynamics of mortality over short time intervals]

Nitric [correction of Nitrous] oxide is most likely a queer "end mediator" giving rise to vasoplegia in septic shock patients. The study is aimed at comparative assessment of kinetic changes in the synthesis of nitric [correction of nitrous] oxide in experimentally induced sepsis model with the corresponding hemodynamic parameters. The laboratory animals--pigs--are divided up in two groups, and exposed to general narcosis induction, orotracheal intubation and mechanical ventilation under controlled regimen. The hemodynamic parameters studied include: MAP, CO and SVR. Additional endotoxin (1 mg/50 ml) in the form of infusion is given to the animals in the sepsis group. Nitrate production mirrors NO synthesis, insofar as there are no other relevant mechanisms of nitrate synthesis. The kinetic parameters of nitrate production are estimated using stable nitrate isotopes--N15. The theory of compartment models and appropriate computerized simulation are used to calculate the respective constants. In the endotoxin treated group (n = 5) a significantly higher level of synthesis of induced NO production is documented--26 +/- 9 mumol/h, as compared to production in the control group--6 +/- 7 mumol/h, as well as a significant increase in cardiac output and systemic vascular resistance reduction. The good correlation between enhanced NO production and hemodynamic response (increase in cardiac output and decrease in systemic vascular resistance) corroborates the validity of the method.     

An autopsied case of multiple system atrophy with remarkable cerebral atrophy

OBJECTIVES: We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, on plasma levels of interleukin (IL) IL-6, IL-8, tumor necrosis factor-alpha (TNFalpha) and nitrite/nitrate (NO2-/ NO3-) in patients with severe septic shock. DESIGN: Prospective clinical study. SETTING: Surgical intensive care unit at a university hospital. PATIENTS: 11 consecutive patients with severe septic shock. INTERVENTIONS: Standard hemodynamic measurements were made and blood samples taken at intervals before, during, and after a 12-h infusion of L-NAME 1 mg x kg(-1) x h(-1) for determination of plasma IL-6, IL-8, TNFalpha and NO2-/NO3- concentration. MEASUREMENTS AND RESULTS: Patients with sepsis had increased plasma levels of IL-6, IL-8, TNFalpha and NO2-/NO3- (p < 0.05). Plasma levels of IL-6. IL-8, and NO2-/NO- were negatively correlated with systemic vascular resistance (r = -0.62, r = -0.65, and r = -0.78, respectively, all p < 0.05). Continuous infusion of L-NAME increased mean arterial pressure and systemic vascular resistance, with a concomitant reduction in cardiac output (all p < 0.01). No significant changes were seen in levels of plasma IL-6, IL-8, and NO-/NO3- during the 24-h observation period. Plasma levels of TNFalpha were significantly reduced during L-NAME infusion compared to baseline (p < 0.05). CONCLUSIONS: NO plays a role in the cardiovascular derangements of human septic shock. Inhibition of NO synthesis with L-NAME does not promote excessive cytokine release in patients with severe sepsis.     

L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.     

Electrophysiological localization of brain regions involved in perceptual memory

In activated macrophages the inducible form of nitric oxide synthase (i-NOS) generates high amounts of toxic mediator, nitric oxide (NO) which contributes to the circulatory failure associated with septic shock. A sesquiterpene lactone compound (yomogin) isolated from medicinal plant Artemisia princeps Pampan inhibited the production of NO in LPS-activated RAW 264.7 cells by suppressing i-NOS enzyme expression. Thus, yomogin may be a useful candidate for the development of new drugs to treat endotoxemia and inflammation accompanied by the overproduction of NO.     

Psychiatric illness and family support in children and adolescents with diabetic ketoacidosis: a controlled study

BACKGROUND: In the adult respiratory distress syndrome, nitric oxide (NO) inhalation improves oxygenation through reducing ventilation-perfusion mismatching, but detailed information on the pulmonary effects of NO inhalation in septic shock is scarce. The present study investigated the effects of inhaled NO on alveolar dead space (Vdalv) and venous admixture as well as on respiratory system compliance (Crs) and respiratory system resistance (Rrs) in a porcine model of septic shock. Protective effects of NO are discussed. METHODS: Thirteen anaesthetised and ventilated pigs were given an infusion of endotoxin for an observation time of 220 min to induce acute lung injury (ALI). In the NO-early group (n=6), an inhalation of 60 ppm NO was started simultaneously with the endotoxin infusion and continued for 190 min. In 7 control/NO-late animals, 60 ppm NO was administered for 30 min following 190 min of endotoxin infusion. Haemodynamics, single-breath CO2-, pressure-, and flow signals were recorded. RESULTS: Endotoxin induced haemoconcentration, pulmonary vasoconstriction, and a decrease in Crs, while venous admixture, Vdalv, and Rrs increased. In the NO-early group, the pulmonary vasoconstriction was attenuated, no increase in pulmonary venous admixture or in Vdalv was seen before cessation of NO, and the improvements in oxygenation outlasted the NO inhalation. In the control/NO-late group, the NO inhalation reversed the changes in dead space and venous admixture. NO had no effect on the changes in respiratory mechanics. CONCLUSION: In porcine ALI, 60 ppm NO diminishes pulmonary vasoconstriction and improves gas exchange by reducing pulmonary venous admixture and alveolar dead space, but does not prevent a fall in Crs. NO inhalation may help prevent long-lasting pulmonary failure.     

Fetal hydrocolpos

This work studies the role that nitric oxide (NO) plays in ischemia/reperfusion (I/R) of the rat kidney. Sprague-Dawley rats, weighing 250-300 g, were subjected to 75 min of warm ischemia and contralateral nephrectomy. The animals were divided into six groups (n = 12 per group): ischemic control (IC) with normal saline, L-NG-mono-methyl-arginine (L-NMMA) 50 mg/kg, L-arginine (L-Arg) 300 mg/kg, Na-nitroprusside (Na-NP) 2.5 mg/kg, the combination of L-NMMA+Na-NP at the doses used before, and the sham group. All animals received the drug intravenously 60 min prior to ischemia. Survival was evaluated at seven days. Renal damage was assessed by kidney function tests (serum creatinine and blood urea nitrogen) and light histology. Lipid peroxidation was measured in renal tissue using the thiobarbituric acid assay. Significantly better survival was seen in the Na-NP group, as compared to the rest of the study. Serum creatinine at 24 and 48 hr showed a significant difference between the Na-NP group and the other groups. Histological damage was minimal in the Na-NP group. Clearly, the Na-NP had the most beneficial effect in survival and histological structure. Lipid peroxidation was significantly different, with the lower levels seen in the L-NMMA group and the higher levels in the Na-NP group. In base to these results, we conclude that exogenous NO has a beneficial and protective effect of the ischemically damaged rat kidney. This protection is independent of lipid peroxidation. Endogenous NO production does not play a role in I/R injury in our model.     

An unknown genetic defect increases venous thrombosis risk, through interaction with protein C deficiency

OBJECTIVES: Nitric oxide (NO), a highly reactive species produced by the activity of NO synthases (NOS), is normally present in the exhaled air of humans and animals. Exhaled NO concentration increases significantly in humans with sepsis and animals, but neither the source nor NOS isoforms responsible for this rise in pulmonary NO production are known. The main objective of this study is to determine the sites and the mechanisms of enhanced NO production in the exhaled air of endotoxemic pigs. DESIGN: Randomized, controlled, animal study. SETTING: University-based animal research facility. SUBJECTS: Thirteen pathogen-free adult female pigs (22 to 27 kg). INTERVENTIONS: Anesthetized pigs were divided into two groups: control and lipopolysaccharides (LPS) (septic) groups. In both groups, extrathoracic (upper airways, nasal, and paranasal) and intrathoracic (bronchi, bronchioles, and alveoli) compartments were ventilated equally with two separate ventilators connected to two tracheal tubes. The LPS group received slow infusion (over 2 h) of Escherichia coli endotoxin (10 microg/kg/h), whereas saline solution was infused into the control group. Expired air of the two compartments was collected throughout the 2-h observation period. The animals were then killed and the lungs were quickly excised and frozen. MEASUREMENTS: Hemodynamic variables were measured in both groups. NO concentration in the exhaled air of both compartments was measured with a chemiluminescence analyser. Pulmonary NOS activity was evaluated by measuring the conversion of L-[2,3H]-arginine to L-[2,3H]-citrulline, and pulmonary expression of NOS was evaluated by immunoblotting. RESULTS: Baseline NO concentration in both groups was significantly higher in the extrathoracic vs intrathoracic compartment (average of 5.2 vs 3.4 parts per billion). Endotoxin infusion elicited a significant and early (after 45 min) rise in exhaled NO concentration in the extrathoracic compartment. Exhaled NO in the intrathoracic compartment also rose significantly but after 90 min of endotoxin infusion. Measurement of lung NOS activity showed a substantial rise in Ca++/calmodulin-dependent activity in the LPS group with no rise in Ca++/calmodulin-independent activity. Immunoblotting of lung tissue samples indicated the absence of the inducible isoform in both groups of animals. Moreover, LPS injection elicited no significant alterations in the pulmonary expression of the endothelial and the neuronal isoforms. CONCLUSIONS: Both extrathoracic and intrathoracic compartments contribute to the rise in exhaled NO production in experimental septic shock. The rise in exhaled NO production is due to increased activity of constitutive NOS isoforms as a result of increased cofactor availability and/or downregulation of the endogenous inhibitors of NOS.     

Role of laparoscopy in the management of chronic pelvic pain

OBJECTIVE: The pharmacokinetics of N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was investigated in patients with septic shock. METHODS: Blood was sampled at intervals before, during and after 12-h infusion of L-NAME 1 mg x kg(-1) x h(-1) in nine septic shock patients for determination of plasma concentrations by high-performance liquid chromatography (HPLC). In three patients the renal clearance of the drug was determined. RESULTS: Incubation of L-NAME with plasma and blood in vitro revealed hydrolysis to N(G)-nitro-L-arginine (L-NOARG), the active inhibitor of NO synthesis. L-NOARG did not undergo further degradation. Continuous intravenous infusion of 1 mg x kg(-1) x h(-1) of L-NAME for 12 h in patients with septic shock increased blood pressure and resulted in increasing plasma concentrations of L-NOARG (Cmax 6.2 microg x ml(-1) at 12 h) whereas L-NAME concentrations reached a plateau within 1.5 h (Cmax 1.0 microg x ml(-1)). After the infusion was stopped L-NAME disappeared from the plasma rapidly (half-life 19.2 min) whereas L-NOARG concentration declined slowly (half-life 22.9 h). The calculated volume of distribution for L-NAME was 0.451 x kg(-1) body weight and 1.961 x kg(-1) for L-NOARG. The renal clearance for L-NOARG was 3.5% of total body clearance for L-NOARG, whereas L-NAME could not be detected in urine. CONCLUSION: We conclude that vasoconstriction with L-NAME in septic patients may result from hydrolysis to L-NOARG, the active inhibitor of NO synthesis. The long plasma half-life and large volume of distribution for L-NOARG suggests extensive distribution to extravascular tissues. Since renal excretion is minimal, elimination of the metabolite L-NOARG follows other pathways.