Effect of propentofylline on free radical generation during cerebral hypoxia in the newborn piglet

The present study tests the hypothesis that propentofylline, an adenosine re-uptake inhibitor, will reduce free radical generation during cerebral hypoxia. Ten newborn piglets were pretreated with propentofylline (10 mg/kg), five of which were subjected to hypoxia, while the other five were maintained at normoxia. Five untreated control piglets underwent the same conditions. Hypoxia was induced through a decrease in FiO2 to 0.11 and documented biochemically by a decrease in ATP and phosphocreatine levels. Free radical formation in the cortex was detected directly using electron spin resonance spectroscopy with a spin trap technique. Results demonstrate that free radicals, corresponding to the alkoxyl radical, increased significantly following hypoxia, and that this increase was inhibited by pretreatment with propentofylline. Conjugated dienes, a lipid peroxidation product, also increased following hypoxia and were subsequently inhibited by propentofylline. The administration of propentofylline also significantly limited the hypoxia-induced decrease in tissue levels of ATP and phosphocreatine. These data demonstrate that pretreatment with propentofylline decreased free radical generation and lipid peroxidation as well as preserved high energy phosphates during cerebral hypoxia.     

Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.     

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

The long-sleep (LS) and short-sleep (SS) mice were selected for differences in sensitivity to ethanol but also differ in response to propofol and some neurosteroids. To determine the role of strychnine-sensitive glycine receptors in genetic differences between these mice, effects of propofol, ethanol and pregnenolone sulfate on glycine responses were compared in Xenopus oocytes expressing mRNA extracted from spinal cord of LS and SS mice. The two lines of mice did not differ in sensitivity to glycine, ethanol or pregnenolone sulfate. However, receptors expressed from LS mRNA were more sensitive to the potentiation induced by propofol than those from SS. Binding of [3H]strychnine to spinal cord membranes demonstrated a similar affinity and density of receptors in LS and SS. These results suggest that glycine receptor function could account for differences in propofol sensitivity between LS and SS mice, but may not be responsible for the differences in behavioral sensitivity to ethanol or steroids previously reported.     

Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.     

Modulation of cocaine- and methamphetamine-induced behavioral sensitization by inhibition of brain nitric oxide synthase

Evidence suggests the existence of multiple interactions between dopamine, glutamate and nitric oxide (NO) in brain structures associated with psychomotor stimulation. The present study was undertaken to investigate the effect of the relatively selective inhibitor of the neuronal nitric oxide synthase (NOS) isoform, 7-nitroindazole (7-NI), on the development of sensitization to the locomotor stimulating effect of cocaine and methamphetamine (METH). Male Swiss Webster mice that received 15 mg/kg cocaine once a day for 5 days developed a marked locomotor sensitization to a challenge cocaine (15 mg/kg) or cross-sensitization to a challenge METH (0.5 mg/kg) injection given after a 10-day drug-free period. This treatment also produced a context-dependent sensitization as evident by the sensitized response to a challenge saline injection. Pretreatment with 7-NI (25 mg/kg) 30 min before cocaine administration (5 days) completely blocked the induction of sensitization to cocaine, the cross-sensitization to METH and the conditioned locomotion induced by cocaine. 7-NI when given alone, either acutely or for 5 days, had no significant effect on the locomotor activity of animals. Animals treated with METH (1.0 mg/kg) for 5 days developed marked sensitization to challenge METH (0.5 mg/kg), cross-sensitization to challenge cocaine (15 mg/kg) and context-dependent locomotion. Pretreatment with 7-NI (25 mg/kg) attenuated the sensitized response to METH and the cross-sensitization to cocaine as revealed after a 10-day drug-free period. However, the METH-induced conditioned locomotion was unaffected by the pretreatment with 7-NI. The present study supports the role of brain NO in the development of sensitization to both psychostimulants, cocaine and METH. However, it appears that the inability of 7-NI to completely abolish the sensitized responses induced after METH administration is the result of the resistible conditioned locomotion caused by METH, but not by cocaine.     

The pulmonary effect of nitric oxide synthase inhibition following endotoxemia in a swine model

OBJECTIVE: To evaluate the pulmonary effect of treatment with N-nitro-L-arginine methyl ester (NAME) with and without inhaled nitric oxide (NO) in a swine model of endotoxemia. DESIGN: Randomized controlled trial. SETTING: Laboratory. INTERVENTIONS: Following a 20-minute intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (200 micrograms/kg), animals were resuscitated with saline solution (1 mL/kg per minute) and observed for 3 hours while mechanically ventilated (fraction of inspired oxygen [FIO2], 0.6; tidal volume, 12 mL/kg; positive end-expiratory pressure, 5 cm H2O). Group 1 (LPS, n = 6) received no additional treatment; group 2 (NAME, n = 5) received NAME (3 mg/kg per hour) for the last 2 hours; group 3 (NO, n = 6) received NAME (3 mg/kg per hour) and inhaled NO (40 ppm) for the last 2 hours; and group 4 (control, n = 5) received only saline solution without LPS. MAIN OUTCOME MEASURES: Cardiopulmonary variables and blood gases were measured serially. The multiple inert gas elimination technique was performed at 3 hours. The wet-to-dry lung weight ratio was measured following necropsy. RESULTS: Administration of LPS resulted in pulmonary arterial hypertension, pulmonary edema, and hypoxemia with increased ventilation perfusion ratio mismatching. None of these changes were attenuated by NAME treatment alone but all were significantly improved by the simultaneous administration of inhaled NO. CONCLUSIONS: Systemic NO synthase inhibition failed to restore hypoxic pulmonary vasoconstriction following LPS administration. The deleterious effects of endotoxemia on pulmonary function can be improved by inhaled NO but not by systemic inhibition of NO synthase.     

Oxygen free radical generation during in-utero hypoxia in the fetal guinea pig brain: the effects of maturity and of magnesium sulfate administration

Previous studies have shown, employing direct measurements with electron spin resonance (ESR) spectroscopy, that hypoxia induces an increased production of oxygen free radicals (OFR) in the brain of the guinea pig fetus. The present study using the same approach, investigated the effects of maturity and Mg2+-pretreatment on hypoxia-induced OFR formation in the guinea pig fetal brain. The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. The control group consisted of term fetuses exposed to normoxia (n=7) and hypoxia (n=7). The experimental groups consisted of the following: (a) for the investigation on maturity effect, preterm fetuses (40 days) exposed to normoxia (n=6) or hypoxia (n=6); and (b) for the Mg2+-pretreatment investigation, term fetuses (60 days) exposed to normoxia (n=6) or hypoxia (n=6) following maternal pretreatment with Mg2+ which consisted of an initial bolus of MgSO4 (600 mg/kg, i.p.) 1 h prior to hypoxia followed by a second dose (300 mg/kg, i.p.). Oxygen free radicals were measured by ESR spectroscopy in the fetal cerebral cortical tissue utilizing phenyl-N-tert-butylnitrone (PBN) spin trapping. Fetal brain tissue hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. In the control group of term fetuses, the cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (71% increase, p<0.01). In the preterm group, the cortical tissue from hypoxic fetuses showed a 33% increase in spin adducts (p<0.001). The baseline free radical generation during normoxia was 22.5% higher at preterm than at term (41.4+/-3.5 units/g issue vs. 33.8+/-9.3 units/g tissue, p<0.05). In Mg2+-treated groups, spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from those of the normoxic group (30.2+/-9.9 units/g tissue, normoxic-Mg2+ vs. 30. 6+/-8.1 units/g tissue, hypoxic-Mg2+). The results indicate that the fetal brain at term may be more susceptible to hypoxia-induced free radical damage than at preterm and that Mg2+ administration significantly decreased the hypoxia-induced increase in oxygen free radical generation in the term fetal guinea pig brain in comparison with non-treated hypoxic group.     

Mechanisms of stroke in sickle cell disease: sickle erythrocytes decrease cerebral blood flow in rats after nitric oxide synthase inhibition

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.     

Immunohistochemical localization of hepatic nitric oxide synthase in normal and transgenic sickle cell mice: the effect of hypoxia

Nitric oxide (NO) generated from L-arginine and molecular oxygen by nitric oxide synthase (NOS) has been shown to influence hepatocellular function and pathology in response to ischemia and certain hepatotoxins. In the present study, we examined the liver of a transgenic line of sickle cell mice for hepatocellular injury and localization of two isoforms of NOS, the endothelial constitutively expressed isoform (EcNOS) and the inducible isoform (iNOS) by immunohistochemistry. Diffuse expression of EcNOS was observed in hepatocytes of control and sickle cell animals maintained under room air conditions. In contrast, iNOS was observed only in the sickle cell mice, well-localized to hepatocytes surrounding the central veins of the lobules. When normal mice were exposed to hypoxic conditions for 4 to 5 days, iNOS immunostaining appeared de novo in a patchy distribution throughout the liver lobules. In the sickle cell mice, hypoxia appeared to increase the subjective intensity of pericentral staining of iNOS. Liver histology was normal in the sickle cell mice maintained under room air conditions, but showed multifocal areas of necrosis when sickling was exacerbated by chronic hypoxic conditions. However, a pericentral zone of preserved architecture was present, corresponding to the region of iNOS staining. We postulate that pericentral induction of iNOS under ambient conditions occurs in transgenic sickle cell mice in response to particularly intense hypoxic conditions near the central veins of the liver. Increases in NO synthesis may occur in this region, which would serve to protect these cells from ischemic damage either directly or by maintaining blood flow. These findings could be relevant to liver pathophysiology in patients with sickle cell disease.     

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.     

Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats

The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L-NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross-sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide.     

Determination of the flux control coefficient of nitric oxide synthase for nitric oxide synthesis in discrete brain regions in vivo

The flux control coefficient of nitric oxide synthase (NOS), for the in vivo synthesis of the key biological mediator nitric oxide (NO), was determined in four rat brain regions with varying NOS activities (cerebral cortex, hippocampus, amygdala and cerebellum) using metabolic control theory. Flux control coefficients were calculated from the ratio of the initial slopes of the fractional effect of the NOS inhibitor N omega-nitro-L-arginine (L-NA) on NO pathway flux and NOS activity. Under conditions of normal behaviour in the rat, NOS had a flux control coefficient not significantly different from one in all regions examined. These data demonstrate that the large majority of flux control for the synthesis of NO in the brain resides in NOS itself and not in the transport of its amino acid precursor, L-arginine, across the blood-brain or neuronal cell membranes. This paper describes the first example in which the control of metabolic flux has been quantified in a mammalian system in vivo and demonstrates the power of metabolic control theory to elucidate the distribution of control within a metabolic pathway in vivo.     

Acute and chronic inhibition of nitric oxide synthase in conscious rabbits: role of nitric oxide in the control of vascular tone

Acute and chronic effects of Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, were examined on the hindquarter hemodynamics of conscious rabbits. After pharmacological autonomic reflex blockade on four experimental days (days 0, 1, 2, and 7), responses to aortic occlusion (balloon cuff, 5-80 s inflation), intra-aortic infusion of acetylcholine, adenosine, and sodium nitroprusside (SNP) were measured before and after vehicle (day 0) or L-NNA (16 mg/kg/h i.v., days 1, 2, and 7). On day 1, L-NNA raised the mean arterial pressure (MAP), and lowered the heart rate (HR) and hindquarter vascular conductance (HVC = abdominal aortic Doppler blood flow/MAP). On days 2 and 7, L-NNA only slowly raised the MAP. The dilator response to acetylcholine was inhibited by L-NNA on day 1 and before and after L-NNA on days 2 and 7. The responses to aortic occlusion, adenosine, or SNP infusion were unaffected by L-NNA treatment on any day. Thus, if nitric oxide synthase inhibition by L-NNA abolishes NO release, then (i) reactive hyperaemia is independent of NO, (ii) basal NO release normalises the arterial pressure in the short term but other factors become important in the long term, and (iii) the blockade by L-NNA of receptor-stimulated NO release by acetylcholine is only very slowly reversible.     

Haemodynamic effects of dopexamine and nitric oxide synthase inhibition in healthy and endotoxaemic sheep

In endothelium-denuded guinea-pig isolated basilar artery preparations, hydroxocobalamin (30, 100 and 300 microM) concentration-dependently inhibited the vasodilator responses to exogenous nitric oxide (NO), whereas the vasodilator responses to nitrergic nerve stimulation were slightly reduced by high (100 and 300 microM) but not by the low (30 microM) concentration of hydroxocobalamin. Vasodilatation in response to sodium nitroprusside (10-100 nM) was totally abolished by 300 microM hydroxocobalamin. In endothelium-intact preparations, vasodilator responses to acetylcholine (0.3-3 microM) were significantly reduced or abolished by hydroxocobalamin (30-300 microM). The mean reduction by hydroxocobalamin of relaxations to acetylcholine was significantly greater than that of the equivalent response evoked by nitrergic nerve stimulation. The findings suggest that the nitrergic transmitter in the guinea-pig basilar artery may be quantitatively less susceptible than the endothelium-derived relaxing factor to the NO scavenger hydroxocobalamin.     

Memory-related changes of nitric oxide synthase activity and nitrite level in rat brain

The changes of nitric oxide synthase (NOS) activity and nitrite level in rat brain regions after spatial learning were investigated. NOS activity was assayed by conversion of [3H]L-arginine to [3H]L-citrulline, and a sensitive fluorometric assay for quantification of nitrite was used. Compared with sham-trained rats, NOS activity and nitrite level in hippocampus and cortex, and also the nitrite level in cerebellum, was elevated significantly one day after rats had learnt a water-rewarded spatial alteration task. These results suggest a spatial memory-related changes of endogenous NO in rat brain, and support the idea that NO participates in learning and memory processes.     

Effects of magnesium sulphate and nitric oxide in pulmonary hypertension induced by hypoxia in newborn piglets

AIM: To examine the haemodynamic effects of intravenous magnesium sulphate on an animal model of neonatal pulmonary hypertension induced by hypoxia. METHODS: The cardiac index (Q), pulmonary arterial pressure (PAP), systemic arterial pressure (SAP), and pulmonary (PVRI) and systemic (SVRI) vascular resistance indices were measured in nine newborn piglets (including three controls). Pulmonary hypertension was induced by lowering the FIO2 to 0.12-0.14, after which there was a significant increase in PAP and PVRI (37% and 142%, respectively; p < 0.01) and a significant fall in SAP and Q (30% and 33%, respectively; p < 0.01). RESULTS: Magnesium sulphate was infused intravenously as four doses of 25 mg/kg, 15 minutes apart, which resulted in a significant mean (SD) increase in serum magnesium (0.83 (0.07) mmol/l to 1.82 (0.19) mmol/l; p < 0.01). After the initial dose SAP, SVRI, PAP and PVRI decreased, but not significantly. Each subsequent dose of (50, 75, 100 mg/kg) was accompanied by further significant reductions in these variables from control baseline (p < 0.05). The PVRI:SVRI ratio remained unchanged throughout. Inhaled nitric oxide (NO) 40 ppm was administered after the last dose of magnesium sulphate. The PVRI:SVRI significantly decreased (p < 0.05), indicating that reversible pulmonary hypertension remained after a maximum dose of magnesium sulphate. CONCLUSIONS: Unlike NO, magnesium sulphate is not a selective pulmonary vasodilator and may lead to deleterious effects on systemic pressures in critically ill newborns.     

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.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.     

Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation

Two low-molecular cytolytic toxins (RmI and RmII) and four trypsin inhibitors were isolated from the aqueous extract of sea anemone Radianthus macrodactylus. The method of isolation involved precipitation with acetone, gel filtration on acrylex P-4, ion-exchange chromatography on CM-32 cellulose, affinity chromatography on trypsin-binding sepharose 4B, ion exchange chromatography on an Ultrapore TSK CM-3SW column, and reversed phase HPLC on a Silasorb C18 column. RmI, RmII, and JnI inhibitor displayed molecular masses 5100, 6100, and 7100 Da, respectively, when subjected to SDS-PAGE. The isoelectric points were 9.2 and 9.3 for RmI and RmII, respectively. The amino acid composition and N-terminal amino acid residue (glycine) were determined for RmI, RmII, and JnI. Both proteins were nontoxic to mice and crabs. Hemolytic activity was determined to be 25 and 20 HU/mg for RmI and RmII, respectively, and their action on erythrocyte membrane was not inhibited by exogenous sphingomyelin. RmI and RmII exhibited antihistamine activity.