Why is folate effective in preventing neural tube closure defects?

Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner.

MAIN CONCLUSION: By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.

Glutaraldehyde cross-linked glutamate oxidase coated microelectrode arrays: selectivity and resting levels of glutamate in the CNS.

Glutaraldehyde is widely used as a cross-linking agent for enzyme immobilization onto microelectrodes. Recent studies and prior reports indicate changes in enzyme activity and selectivity with certain glutaraldehyde cross-linking procedures that may jeopardize the performance of microelectrode recordings and lead to falsely elevated responses in biological systems. In this study, the sensitivity of glutaraldehyde cross-linked glutamate oxidase-based microelectrode arrays to 22 amino acids was tested and compared to glutamate. As expected, responses to electroactive amino acids (Cys, Tyr, Trp) were detected at both nonenzyme-coated and enzyme-coated microelectrodes sites, while the remaining amino acids yielded no detectable responses. Electroactive amino acids were effectively blocked with a m-phenylene diamine (mPD) layer and, subsequently, no responses were detected. Preliminary results on the use of poly(ethylene glycol) diglycidyl ether (PEGDE) as a potentially more reliable cross-linking agent for the immobilization of glutamate oxidase onto ceramic-based microelectrode arrays are reported and show no significant advantages over glutaraldehyde as we observe comparable selectivities and responses. These results support that glutaraldehyde-cross-linked glutamate oxidase retains sufficient enzyme specificity for accurate in vivo brain measures of tonic and phasic glutamate levels when immobilized using specific "wet" coating procedures.

Methyl jasmonate-induced defense responses are associated with elevation of 1-aminocyclopropane-1-carboxylate oxidase in Lycopersicon esculentum fruit.

It has been known that methyl jasmonate (MeJA) interacts with ethylene to elicit resistance. In green mature tomato fruits (Lycopersicon esculentum cv. Lichun), 0.02mM MeJA increased the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), and consequently influenced the last step of ethylene biosynthesis. Fruits treated with a combination of 0.02 MeJA and 0.02 α-aminoisobutyric acid (AIB, a competitive inhibitor of ACO) exhibited a lower ethylene production comparing to that by 0.02mM MeJA alone. The increased activities of defense enzymes and subsequent control of disease incidence caused by Botrytis cinerea with 0.2mM MeJA treatment was impaired by AIB as well. A close relationship (P<0.05) was found between the activity alterations of ACO and that of chitinase (CHI) and ß-1,3-glucanase (GLU). In addition, this study further detected the changes of gene expressions and enzyme kinetics of ACO to different concentrations of MeJA. LeACO1 was found the principal member from the ACO gene family to respond to MeJA. Accumulation of LeACO1/3/4 transcripts followed the concentration pattern of MeJA treatments, where the largest elevations were reached by 0.2mM. For kinetic analysis, K(m) values of ACO stepped up during the experiment and reached the maximums at 0.2mM MeJA with ascending concentrations of treatments. V(max) exhibited a gradual increase from 3h to 24h, and the largest induction appeared with 1.0mM MeJA. The results suggested that ACO is involved in MeJA-induced resistance in tomato, and the concentration influence of MeJA on ACO was attributable to the variation of gene transcripts and enzymatic properties.

Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment.

We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli.

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.

Cytotoxicity evaluation after coexposure to perchloroethylene and selected peroxidant drugs in rat hepatocytes.

There is evidence of hepatotoxic effects caused by Perchloroethylene (PCE), presumably due to reactive metabolic intermediates; lipid peroxidation is under study as a potential mechanism of toxicity. We aimed to verify if PCE levels comparable to those reached in the blood of exposed subjects can cause cell damage and lipid peroxidation. The association of PCE with lipid peroxidation inducing drugs (cyclosporine A, valproic acid and amiodarone) was also tested on rat isolated hepatocytes. AST and LDH release, MTT test and lipid peroxidation assay showed that PCE determines dose-dependent effects on rat isolated hepatocytes. The toxic potential resulting from our data would be valproic acid < cyclosporine A < amiodarone. While valproic acid and cyclosporine caused a mild toxicity, the effects of amiodarone were more severe; in particular, the association of PCE with amiodarone showed a clear additive effect. The role of lipid peroxidation in the liver toxicity exerted by the tested compounds was confirmed by our data, and resulted relevant after treatment of cells with amiodarone and PCE. Extrapolating these results to human, we can suggest that a subject professionally exposed to PCE, who chronically assumes a lipid peroxidation inducing drug like amiodarone, may be potentially exposed to a higher risk of liver toxicity.

Salt-induced expression of NADP-dependent isocitrate dehydrogenase and ferredoxin-dependent glutamate synthase in Mesembryanthemum crystallinum.

NADP-specific isocitrate dehydrogenase is a key cytosolic enzyme that links C and N metabolism by supplying C skeletons for primary N assimilation in plants. We report the characterization of the transcript Mc-ICDH1 encoding an NADP-dependent isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) from the facultative halophyte Mesembryanthemum crystallinum L., focussing on salt-dependent regulation of the enzyme. The activity of NADP-ICDH in plants adapted to high salinity increased in leaves and decreased in roots. By transcript analyses and Western-type hybridizations, expression of Mc-ICDH1 was found to be stimulated in leaves in salt-adapted M. crystallinum. By immunocytological analyses, NADP-ICDH proteins were localized to most cell types with strongest expression in epidermal cells and in the vascular tissue. In leaves of salt-adapted plants, signal intensities increased in mesophyll cells. In contrast to Mc-ICDH1, the activity and transcript abundance of ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1), which is the key enzyme of N assimilation and biosynthesis of amino acids, decreased in leaves in response to salt stress. The physiological roles of NADP-ICDH and Fd-GOGAT in the adaptation of plants to high salinity are discussed.

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at

Differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls in response to various stimuli.

Ethylene can be produced by a variety of developmental and environmental factors such as ripening, the plant hormone auxin, and mechanical wounding via a biosynthetic pathway including AdoMet synthase, ACC synthase, and ACC oxidase steps. ACC synthase and ACC oxidase are known to be encoded by multigene families, and are believed to be differentially expressed in response to various stimuli. In mung bean, ACC synthase is encoded by 7 genes, ACS1, ACS2 ACS3, ACS4, ACS5, ACS6, and ACS7, and ACC oxidase by 2 genes, ACO1 and ACO2. In this study, was have investigated differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls under various conditions by the semiquantitative RT-PCR method. Primers which can specifically bind and amplify each cDNAs of ACS1, ACS2, ACS3, ACS4, ACS5, ACS6, ACS7, and ACO1, and ACO2 were designed and used to monitor the responses to various stimuli. Transcripts of ACO1 and ACO2 were accumulated constitutively in the hypocotyl segments even without andy treatment. After cold treatment on intact plant, transcripts of ACS5, ACS6, and ACS7 were accumulated in the hypocotyl segments. We also found the excision of hypocotyl segments and incubation in a buffer solution, a typical way of chemical treatments to hypocotyl segments, lowered the level of ACO2 transcripts with little change of the level of ACO1 transcripts. In response to incubation with IAA (0.1 mM) of excised hypocotyl segments, transcripts of ACS1, ACS6, and ACS7 were accumulated and the level of ACO2 transcripts was increased. Transcripts of ACS1, ACS2, ACS3, ACS5, ACS6 and ACS7 were induced by incubation with OGA (50 micrograms/ml), while the transcripts of ACS4 were accumulated and the level of ACO2 transcripts was increased by incubation with 1 mM LiCl. Our results strongly suggest that all seven ACC synthase genes and two ACC oxidase genes must be active and each gene is differentially regulated by a different subset of the inducing factors.

The requirements for Ca2+, protein phosphorylation, and dephosphorylation for ethylene signal transduction in Pisum sativum L.

The role of Ca2+ and protein phosphorylation in the transduction of the ethylene signal resulting in induction of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase has been studied in peas (Pisum sativum L.) by a pharmacological approach. 2,5-Norbornadiene (NBD) and aminoethoxyvinylglycine (AVG) reduced the basal level of ACC oxidase transcript and its enzyme activity. Only NBD was shown to inhibit the ethylene response, the accumulation of ACC oxidase transcript and the stimulation of its enzyme activity. Ethylene influenced 45Ca2+ influx into the segment tissues from pea epicotyls, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N'N'-tetraacetic acid (EGTA) a Ca2+ chelator, inhibited the ethylene response. Ca2+ depletion by pretreatment with EGTA also blocked the ethylene response, which almost completely recovered when Ca2+ was added exogenously after Ca2+ depletion. Ca2+ channel blockers, verapamil, and LaCl3, used to certify the role of extracellular Ca2+, all inhibited the ethylene response. A protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), and protein phosphatase inhibitors, vanadate and okadaic acid, also inhibited the ethylene response. The results of the present study suggest that Ca2+ influx from the extracellular space, protein phosphorylation, and dephosphorylation are required for the induction of ACC oxidase by ethylene.

Peroxisomal enzyme activity changes in the tail of anuran tadpoles during metamorphosis.

This study attempts to clarify peroxisomal enzyme activity changes in the atrophying tail of anuran tadpoles. Changes in catalase, D-amino acid oxidase and urate oxidase activity were spectrophotometrically investigated using tadpole tails of Rana japonica and Rana nigromaculata. In R. japonica, total catalase activity decreased in tails undergoing regression during spontaneous and DL-thyroxine (T4)-induced metamorphosis, whereas total D-amino acid oxidase and urate oxidase activity increased. In R. nigromaculata, total activity of catalase decreased in tails regressing spontaneously. Total D-amino acid oxidase activity increased during advanced stages of tail regression, but total urate oxidase activity decreased. Specific activity of tadpole peroxisomal enzymes in the above two species was found to be highest for D-amino acid oxidase, followed by urate oxidase and then catalase at latter stages of normal tail regression. Atrophying tadpole tails develop a mechanism for hydrogen peroxide production, which may contribute to cell death in this organ.

Exogenous nitric oxide regulates IFN-gamma plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages.

This study was performed to determine the effects of nitric oxide (NO) on the expression of inducible NO synthase (iNOS) in mouse macrophages. We used the NO donor diethylamine dinitric oxide (DEA/NO) and the mouse macrophage cell line ANA-1 in these experiments. ANA-1 macrophages did not express iNOS mRNA either constitutively or following exposure to 100 U/ml IFN-gamma alone, to 10 ng/ml LPS alone, or to 200 microM DEA/NO alone. Similarly, ANA-1 macrophages did not express detectable levels of iNOS mRNA following treatment with 100 U/ml IFN-gamma plus 200 microM DEA/NO. However, IFN-gamma (100 U/ml) plus LPS (10 ng/ml) induced high levels of iNOS mRNA in ANA-1 macrophages after 6 h. Low concentrations of DEA/NO (approximately 1 to 12 microM) caused up to a 2.5-fold augmentation of IFN-gamma plus LPS-induced iNOS mRNA expression. In contrast, 200 microM DEA/NO suppressed IFN-gamma plus LPS-induced iNOS mRNA expression (60% decrease). The effects of DEA/NO were gene-specific because DEA/NO did not affect the IFN-gamma plus LPS-induced expression of TNF-alpha mRNA. Moreover, the biphasic effects of DEA/NO were specifically due to released NO. Diethylamine and nitrite were unable to regulate IFN-gamma plus LPS-induced gene expression in ANA-1 macrophages. Time-response experiments suggested that the effects of NO were short-lived and occurred early during the induction of iNOS gene expression. The effects of NO were not limited to iNOS mRNA expression but were apparent at the level of iNOS protein expression and enzymatic activity. Overall, these results suggest that NO has immunoregulatory effects and may control the extent and duration of cytokine- and/or endotoxin-induced iNOS expression in macrophages.

Nitric oxide synthase activity is inducible in rat, but not rabbit alveolar macrophages, with a concomitant reduction in arginase activity.

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 x 10(6) cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of 3H-citrulline (NO synthase activity) and 3H-ornithine (arginase activity) were determined. During incubation of rat alveolar macrophages with 3H-arginine clear amounts of 3H-citrulline and 3H-ornithine (3.8 and 4.6% of the added 3H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for 3H-citrulline and 3H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of 3H-citrulline was increased about 30-fold and this was accompanied by a reduction in 3H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mumol/l). Inhibition of NO synthase by NG-monomethyl-L-arginine (L-NMMA, 100 mumol/l) in LPS treated alveolar macrophages reduced the formation 3H-citrulline by more than 90% and restored the 3H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of 3H-citrulline and 3H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhalational anesthetics do not alter nitric oxide synthase activity.

Inhalational anesthetics inhibit the nitric oxide (NO)-soluble guanylate cyclase signaling pathway in vascular and neuronal tissues and it has been proposed that this inhibition is due to several mechanisms, which include a direct inhibition of NO synthase. To determine the direct interaction of anesthetics with NO synthase, the effects of halothane, isoflurane and enflurane on NO synthase activity of bovine and rat brains and cultured bovine aortic endothelial cells were investigated. Halothane and enflurane at 1% to 3% concentrations produced no significant effect on crude bovine brain NO synthase activity, as measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Similarly, crude rat brain NO synthase activity was not affected by exposure to 1% to 4% halothane or isoflurane. The effects of inhalational anesthetics on the crude bovine brain NO synthase activity were not altered when assayed at two different temperatures (22 degrees C and 37 degrees C). Halothane and isoflurane produced no significant effects on the activity of partially purified rat brain NO synthase at different concentrations of L-[3H]arginine in the reaction mixture. Partially purified endothelial NO synthase, when equilibrated with halothane or isoflurane (0.5-2%), exhibited no significant alteration in enzyme activity. This study suggests that the effects of inhalational anesthetics on NO synthesis in rat and bovine brains and in vascular endothelial cells are not due to their direct interaction with NO synthase.

Effects of nitric oxide (NO) synthesis inhibition on the development of supersensitivity to stereotypy and locomotion stimulating effects of methamphetamine.

The present study examined the effects of nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (LNAME; 30 and 60 mg/kg, i.p.) on the development of supersensitivity to stereotypy as well as locomotion stimulating effects of methamphetamine (MA) (3.22 and 0.805 mg free base/kg, s.c., respectively). Rats treated with MA for 10 days showed enhancement in MA-induced stereotypy and locomotor activity. Rats pretreated with LNAME prior to MA also showed enhancement in the two types of behavior, also they showed significantly reduced stereotypy scores compared to those treated with MA alone. The results suggest that NO synthesis is not critically involved in the development of behavioral supersensitivity to stereotypy stimulating as well as locomotion stimulating effect of MA. However, No synthesis may have a modulatory role in behavioral sensitization in stereotypy.

Nitric oxide synthase inhibited by audouine in the rat brain.

Nitric oxide synthase (NOS) synthesizes nitric oxide (NO) from L-arginine (Arg) which has a guanidino group in its molecule. Audouine, a derivative of Arg, is the diguanidino compound. In this study, the effects of audouine on rat brain NOS activity were investigated by measuring nitrite and nitrate formation. Audouine inhibited NOS activity in a competitive (Ki = 2.10 microM) and partially uncompetitive (Ki = 49.7 microM) manner. Audouine is not substituted at the guanidino nitrogen, in contrast to most previously reported NOS inhibitors which were synthesized by substituting the guanidino nitrogen of Arg. Audouine is a novel inhibitor of NOS and should be useful for investigating the chemical nature of NOS and the roles of NO in the central nervous system.

Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.

N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids.

1. Lipopolysaccharide (LPS) co-induces nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) in J774.2 macrophages. Here we have used LPS-activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX-2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX-2 activity was assessed by measuring the formation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Western blot analysis was used to determine the expression of COX-2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS-treated rats. 2. Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 microgram ml-1) for 24 h caused a time-dependent accumulation of nitrite and 6-keto-PGF1 alpha in the cell culture medium which was first significant after 6 h. The formation of both 6-keto-PGF1 alpha and nitrite elicited by LPS was inhibited by cycloheximide (1 microM) or dexamethasone (1 microM). Western blot analysis showed that J774.2 macrophages contained COX-2 protein after LPS administration, whereas untreated cells contained no COX-2. 3. The accumulation of 6-keto-PGF1 alpha in the medium of LPS-activated J774.2 macrophages was concentration-dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1-1000 microM). Sodium nitroprusside (1-1000 microM) also acutely (30 min) inhibited COX-2 activity in broken cell preparations of LPS-activated (12 h) J774.2 macrophages, in a similar concentration dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX-2 in broken cell preparations. In the presence of these co-factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 microg ml-1) and NG-monomethyl-L-arginine (L-NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX-2 in cell homogenates with co-factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 microg ml-1)and L-NMMA (1 mM), the addition of SNP (0.001-1I000 microM) increased in a concentration-dependent manner the accumulation of 6-keto-PGF1a in intact cells (measured at 24 h) and COX-2 activity in cell homogenates in the presence of co-factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h)decreased the amount of COX-2 protein induced by LPS in J774.2 macrophages.4. Indomethacin (30 1AM) abolished the formation of 6-keto-PGFa by LPS-activated macrophages, but had no effect on the release of nitrite. Conversely, L-NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6-keto-PGFIa an effect which was reversed by excess L-arginine (3 mM)but not by D-arginine. Similarly, the decrease in nitrite formation caused by L-NMMA was partially reversed by L-arginine (3 mM), but not by D-arginine. L-NMMA (10 mM; together with LPS for 12 h)increased the amount of COX-2 protein induced by LPS in J774.2 macrophages.5. In separate experiments, J774.2 macrophages were activated with LPS (1 microg ml-1), and L-NMMA(10 mM) was added for various times (0.5-24 h) before the collection of mediun at 24 h. L-NMMAenhanced the release of 6-keto-PGFI,, in a time-dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h. 6. In experiments on male Wistar rats, we investigated the effect of L-NMMA on the release of prostanoids (6-keto-PGF1a prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA,30nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS-treated rats was unaffected by L-NMMA intraperitoneally (30 mg kg-1) for 6 h together with LPS(5 mg kg-1) or LPS vehicle. Similarly, acute (5 min) in vitro exposure to L-NMMA (1 mM) of the perfused organs from control and LPS-treated animals did not change the release of prostanoids elicited by AA (30 nmol).7. These results show that LPS causes the induction of iNOS and COX-2 in J774.2 macrophages. The co-release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag-time of 6-12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX-2 activity and induction. These results demonstrate that NOS and COX can be co-induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity.In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX-2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX.