Sensitive quantitation of nitric oxide by EPR spectroscopy

A recent method for NO detection is electron paramagnetic resonance (EPR) with ferrous and mononitrosyl dithiocarbamate (Fe2+ (DETC)2) for spin trapping [Menon, N.K., et al., J.Mol. Cell Cardiol., 23:389; 1991]. However, by this technique, we failed to detect the spectrum of the NOFe2+ (DETC)2 complex in biological systems because of the low solubility of Fe2+ (DETC)2 and rapid oxidation of NOFe2+ (DETC)2 complex. To overcome these problems, we modified this method by adding albumin to solubilize Fe2+ (DETC)2 and Na2S2O4 as a strong reductant to increase the sensitivity and stability of the EPR spectrum of the NOFe2+ (DETC)2 complex. The optimal concentrations of these reagents were 3.3 mM of Fe2+ and DETC, 33 mg/ml albumin and 2 M Na2S2O4. The detection limit was less than 10 pmol/ml under these conditions. By this modified method, we succeeded in quantifying NO production from porcine aorta induced by forskolin.     

18O exchange in suspensions of red blood cells: determination of parameters of mass spectrometer inlet system

A variety of anatomic and functional neuroimaging findings are associated with Alzheimer's disease (AD). One of the strongest imaging associations identified is between AD and hippocampal atrophy. The epsilon4 allele of the apolipoprotein E (ApoE) gene increases the risk of developing AD and lowers the mean age of onset of the disease. The purpose of this study was to assess the association between hippocampal volume and ApoE polymorphisms in elderly control subjects and in patients with probable AD. We performed magnetic resonance imaging-based volume measurements of the hippocampus in 125 cognitively normal elderly controls and 62 patients with probable AD. ApoE genotyping was performed by using standard methods. Hippocampal volumes were significantly smaller in AD cases than in control subjects. Hippocampal volumes did not differ significantly within either clinical group on the basis of ApoE genotype. Both the epsilon4 allele of ApoE and hippocampal atrophy were significantly but independently associated with AD.     

Electrochemical nitric oxide and nitrogen dioxide analyzer for use with inhaled nitric oxide

The use of inhaled nitric oxide (NO) in research and clinical applications requires the monitoring of NO and its autooxidation product nitrogen dioxide (NO2) in inspired gas and in the ambient environment. We describe an inexpensive electrochemical NO and NO2 analyzer that uses a critical orifice constant-flow controller and a microprocessor crossover correction for the measurement of NO and NO2 in the concentration range relevant to the use of inhaled NO. The analyzer proved to have good accuracy and precision for NO and NO2 in the range of concentrations relevant to studies of inhaled NO. In this range, the performance was similar to that of a chemiluminescence analyzer, and the response characteristics were not affected by varying the O2 concentration of the mixtures analyzed.     

Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems

Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant neoplastic disorder causing central nervous system haemangioblastomas. The VHL gene (3p25-3p26) is known to be a tumour suppressor gene, with its inactivation being responsible for a predisposition to tumour development. As far as we know, the present report of VHL disease manifestation in identical twins is unique. Genetic inquiry into the family background did not reveal this disease among their progenitors. For presymptomatic diagnosis of 17 presently unaffected family members, constitutional DNA of the twins was screened for VHL germline mutations, using loss of heterozygosity studies and exon-specific DNA sequencing. To determine the influence of somatic mutations of the VHL gene in tumourigenesis, DNA of five surgically removed intracerebral haemangioblastomas of the identical twins was analyzed in comparison with their constitutional DNA by DNA sequencing of the complete VHL coding region. However, no allelic losses were found for the VHL gene or for various other tumour suppressor genes (p53, BRCA1, BRCA2, DCC, and MCC). Furthermore, no mutations were found in the constitutional DNA of either twin sister or in the DNA of all five tumour lesions. Based on our observations, we conclude that in certain VHL families, presymptomatic molecular diagnosis of the disease is not feasible and requires close clinical surveillance of all individuals at risk.     

Impact of surgery on nitric oxide in rats: evidence for activation of inducible nitric oxide synthase

We investigated the effect of euvolemic surgical preparation, on chemical indices of activity of the nitric oxide (NO) system, in anesthetized, acutely prepared rats. The urinary excretion of NO2+NO3 (UNOXV) and cGMP (UcGMPV) increased progressively during the experiment. Pretreatment with aminoguanidine or dexamethasone, inhibitors of inducible NO synthase (iNOS), prevented the increase in UNOXV and UcGMPV but had no impact on mean arterial pressure (BP), renal vascular resistance (RVR) or GFR. Since these variables did not change in the conscious rat, the increased UNOXV results from some aspect of the acute surgical preparation. When acutely prepared rats received L-NAME, a non-specific NOS inhibitor, BP and RVR increased but paradoxical increases in UNOXV and UcGMPV were also seen. Nonselective NOS inhibition (+L-NAME) was fatal in 50% of acutely prepared rats, causing cardiac contracture. The same dose of L-NAME produced no deaths in either conscious chronically catheterized rats or in acutely prepared rats, previously subjected to sterile surgery and acute L-NAME in the conscious state. These data indicate that acute, nonsterile surgery induces expression of iNOS, but that the additional NO generated has no obvious cardiovascular/renal actions. Acute UNOXV and UcGMPV do not predict total NO production, or "hemodynamically active" NO. Generalized NO inhibition in rats acutely stressed by surgery/anesthesia can be fatal.     

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC-7) and 2,2'-(hydroxynitrosoydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10(-5) M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.     

一种四极质谱仪供电控制系统的研制

在四极质谱仪中,各个模块单元所需供电电源的类型和带载能力均不同,同时各模块单元的供电电源还需要实时控制和逻辑顺序控制,而现有仪器的供电系统,一般采用串口或网口实现通讯控制,这两种总线在抗干扰性、实时性和可扩展性方面较差。基于微控制器(ARM)和控制器局域网(CAN)总线,研制了一种新型的四极质谱仪供电控制系统。ARM负责电源的数字控制输出和电源状态监测,CAN总线负责与上位机通讯和模块单元间的通讯。将本系统应用于电感耦合等离子体质谱仪(ICP-MS),进行了背景噪声、短期稳定性和长期稳定性测定试验,测定结果符合国家计量技术规范-四极杆电感耦合等离子体质谱仪校准规范JJF 1159—2006中的要求。     

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity

The effects of substrate, L-Arg and cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the R/S ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 microM H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the Kd values for ligand I was decreased down to 5.9. In the presence of both 1 mM L-Arg and 0.1 mM H4B, the S/R ratios for ligands I and II and the R/S ratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.     

Endothelin and nitric oxide

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide, whereas nitric oxide (NO) is a potent endothelium-derived vasorelaxing factor synthesized from L-arginine by NO synthase (NOS). ET-1 and NO exert many biological actions in a counter-regulatory manner in vascular remodeling and vascular tonus. Furthermore, there exists a close interaction between ET-1 and NO:ET-1 stimulates NO generation, while NO down-regulates the expression of ET-1 gene.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide

Nitric oxide (NO) is produced by a variety of cells within the respiratory tract, including inflammatory epithelial cells. NO has been detected in the exhaled air of normal human subjects, and its concentration is raised in asthmatic patients. To study whether exhaled NO arises from the respiratory tract, we administered a NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA), by inhalation (490 mg) in a double-blind randomized manner in nine normal and six asthmatic subjects. Because exhaled NO may arise from an inducible isoform of NO synthase that may be inhibited by glucocorticosteroids, we also studied the effects of oral prednisolone (30 mg orally for 3 d) in seven normal and six asthmatic subjects in a separate double-blind crossover study with matched placebo. After nebulized L-NMMA, there was a significant fall in peak exhaled NO compared with saline control values, with a mean fall of 43.6 +/- 5.6% in normal subjects (p < 0.01) and of 39.7 +/- 6.5% (p < 0.01) in asthmatic subjects, which persisted for 4 h. There were no effects of L-NMMA inhalation on heart rate, blood pressure, or FEV1 in either normal or asthmatic patients. Administration of oral prednisolone (30 mg) resulted in a fall in exhaled NO concentrations in asthmatic subjects by 21.6 +/- 5.0% at 48 h (p < 0.01) but no significant change in normal subjects. These data suggest that NOS inhibitors may be safely given in normal and asthmatic patients and that the increased exhaled NO seen in asthmatic patients is likely to be caused by induction of inducible NOS.     

Procedures for tandem mass spectrometry on an ion trap storage/reflectron time-of-flight mass spectrometer

In this work, we demonstrate tandem mass spectrometry on an ion trap storage-reflectron time-of-flight mass spectrometer (IT/reTOFMS). Ion isolation and activation were achieved by resonant excitation using multi- and single-frequency waveforms generated from an analog circuit. Product-ion spectra of small polypeptides are obtained, which are comparable in fragmentation to those acquired on sector or hybrid mass spectrometers. Several important parameters governing the tandem mass spectrometry process, including the activation tickle voltage, type of collision gas, activation period and cooling period after the fragmentation were optimized using leucine-enkephalin as a model. Although the limited energy deposition from collisional activation in our experiments does not allow efficient fragmentation of large singly charged polypeptides with m/z higher than 1000, the problem may be partially solved by taking advantage of fragmenting the multiply charged ions produced from the electrospray ionization source as demonstrated for a synthetic polypeptide of molecular weight 2782. Compared to the singly charged form, the reduced m/z of multiply charged forms experience a greater trapping force as described by the pseudopotential well-depth model. Increased pseudopotential well-depths for multiply charged species permit the use of greater fragmentation energy at lower RF potentials. These conditions facilitate the fragmentation of large polypeptides, yet are suitable for trapping singly charged fragments. These experiments indicate that the high efficiency associated with ion dissociation and fragment-ion collection in the trap and the storage capability for detection of ions using the non-scanning mode of the IT/ reTOF analyzer may provide an alternative means for acquiring sequence-specific information of polypeptides at low picomol levels of sensitivity.     

Influence of atmospheric nitric oxide concentration on the measurement of nitric oxide in exhaled air

BACKGROUND: Measurement of nitric oxide (NO) in exhaled air shows promise as a non-invasive method of detecting lung inflammation. However, variable concentrations of NO are measured in environmental air. The aim of this study was to verify a possible relationship between exhaled NO and atmospheric NO values during high atmospheric NO days. METHOD: Exhaled air from 78 healthy non-smokers of mean age 35.3 years was examined for the presence of NO using a chemiluminescence NO analyser and NO levels were expressed as part per billion (ppb). The exhaled air from all the subjects was collected into a single bag and into two sequential bags. Before each test atmospheric NO was measured. RESULTS: The mean (SE) concentration of exhaled NO collected into the single bag was 17.1 (0.6) ppb while the mean values of exhaled NO in bags 1 and 2 were 16.7 (1.3) ppb and 13.8 (1.2) ppb, respectively. The atmospheric NO concentrations registered before each test varied from 0.4 to 71 ppb. There was a significant correlation between exhaled NO in the single bag and atmospheric NO (r = 0.38, p = 0.001). The atmospheric NO concentration also correlated with exhaled NO both in bag 1 (r = 0.44, p = 0.0001) and in bag 2 (r = 0.42, p = 0.0001). These correlations disappeared with atmospheric NO concentrations lower than 35 ppb. CONCLUSIONS: These results indicate a relationship between atmospheric NO and NO levels measured in exhaled air, therefore exhaled NO should not be measured on very high atmospheric NO days.     

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.     

四极杆电感耦合等离子体质谱仪检出限的不确定度评定

依据JJF1159-2006《四极杆电感耦合等离子体质谱仪校准规范》,使用标准溶液GBW(E)130242(Be, In, Bi混合溶液)校准了四极杆电感耦合等离子体质谱仪的检出限,并对其可能引入的不确定度分量（空白溶液测量列的标准偏差、测量重复性、标准溶液的定值以及仪器的分辨力等）进行了分析,建立了数学模型,最后以合成标准不确定度乘以95%置信概率下的扩展因子2获得检出限的扩展不确定度。     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

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.     

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.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.